Methods and unit dose formulations for the inhalation administration of aminoglycoside antibiotics

ABSTRACT

A patient suffering from an endobronchial infection is treated by administering to the patient for inhalation a dose of less than about 4.0 ml of a nebulized aerosol formulation comprising from about 60 to about 200 mg/ml of an aminoglycoside antibiotic, such as tobramycin, in a physiologically acceptable carrier in a time period of less than about 10 minutes. Unit dose devices for storage and delivery of the aminoglycoside antibiotic formulations are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/292,234, filed May 18, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to new and improved unit dosecontainers of aminoglycoside antibiotics, such as tobramycin, fordelivery by aerosol inhalation, and to improved methods of treatment ofsusceptible acute or chronic endobronchial infections.

BACKGROUND OF THE INVENTION

[0003] Progressive pulmonary disease is the cause of death in over 90%of cystic fibrosis (CF) patients (Koch C. et al., “Pathogenesis ofcystic fibrosis,” Lancet 341(8852):1065-9 (1993); Konstan M. W. et al.,“Infection and inflammation of the lung in cystic fibrosis,” Davis P B,ed., Lung Biology in Health and Disease, Vol. 64. New York, N.Y.:Dekker: 219-76 (1993)). Pseudomonas aeruginosa is the most significantpathogen in CF lung disease. Over 80% of CF patients eventually becomecolonized with P. aeruginosa (Fitzsimmons S. C., “The changingepidemiology of cystic fibrosis,” J Pediatr 122(1):1-9 (1993)). Thestandard therapy for P. aeruginosa endobronchial infections is 14 to 21days of parenteral antipseudomonal antibiotics, typically including anaminoglycoside. However, parenteral aminoglycosides, as highly polaragents, penetrate poorly into the endobronchial space. To obtainadequate drug concentrations at the site of infection with parenteraladministration, serum levels approaching those associated with nephro-,vestibule-, and oto-toxicity are required (“American Academy ofOtolaryngology. Guide for the evaluation of hearing handicap,” JAMA241(19):2055-9 (1979); Brummett R. E., “Drug-induced ototoxicity,” Drugs19:412-28 (1980)).

[0004] Aerosolized administration of aminoglycosides offers anattractive alternative, delivering high concentrations of antibioticdirectly to the site of infection in the endobronchial space whileminimizing systemic bioavailability (Touw D. J. et al., “Inhalation ofantibiotics in cystic fibrosis,” Eur Respir J 8:1594-604 (1995);Rosenfeld M. et al., “Aerosolized antibiotics for bacterial lower airwayinfections: principles, efficacy, and pitfalls,” Clinical PulmonaryMedicine 4(2): 101-12 (1997)).

[0005] Tobramycin is commonly prescribed for the treatment of serious P.aeruginosa infections. It is an aminoglycoside antibiotic produced bythe actinomycete, Streptomyces tenebrarius. Low concentrations oftobramycin (<4 μg/mL) are effective in inhibiting the growth of manyGram-negative bacteria and under certain conditions may be bactericidal(Neu, H. C., “Tobramycin: an overview,” J Infect Dis 134, Suppl: S3-19(1976)). Tobramycin is poorly absorbed across mucosal surfaces,conventionally necessitating parenteral administration. Tobramycinactivity is inhibited by purulent sputum: high concentrations ofdivalent cations, acidic conditions, increased ionic strength andmacromolecules that bind the drug all inhibit tobramycin in thisenvironment. It is estimated that 5 to 10 times higher concentrations oftobramycin are required in the sputum to overcome these inhibitoryeffects (Levy J. et al., “Bioactivity of gentamicin in purulent sputumfrom patients with cystic fibrosis or bronchiectasis: comparison withactivity in serum,” J Infect Dis 148(6):1069-76 (1983)).

[0006] Delivery of the poorly absorbed antibiotic tobramycin to theairway by the aerosol route of cystic fibrosis (CF) patients has beendocumented using the aerosol route. Much of this work has been donetoward treatment of chronic lung infections with P. aeruginosa in cysticfibrosis (CF) patients. A multicenter, double blind, placebo-controlled,crossover trial of 600 mg tid of aerosolized tobramycin forendobronchial infections due to P. aeruginosa in 71 CF patientsdemonstrated a significant reduction in sputum density of this pathogenas well as improved spirometry in the treatment group. Emergence of P.aeruginosa strains highly resistant to tobramycin (defined as MIC≧128μg/mL) was comparable in the placebo and treatment groups. The presencein the sputum of Gram-negative organisms other than P. aeruginosaintrinsically resistant to tobramycin occurred with equal frequencyduring administration of tobramycin or placebo (Ramsey B. et al.,“Response to Letter to the Editor: Aerosolized tobramycin in patientswith cystic fibrosis,” N Engl J Med 329:1660 (1993)).

[0007] Although this regimen was found to be both safe and efficacious,it is costly and inconvenient. A survey of the MICs for P. aeruginosaisolates from initial sputum cultures for patients at the Children'sHospital CF Center, Seattle, Wash., in 1993 found that 90% of isolateshad MICs ≦16 μg/mL and 98% of all isolates had MICs ≦128 μg/mL. Thissurvey suggested that achieving a sputum tobramycin concentration of 128μg/mL should treat the endobronchial infection in CF patients (Levy J.et al., “Bioactivity of gentamicin in purulent sputum from patients withcystic fibrosis or bronchiectasis: comparison with activity in serum,” JInfect Dis 148(6):1069-76 (1983)).

[0008] A randomized, crossover study compared the ability of severalnebulizers to deliver tobramycin by measuring peak sputum tobramycinconcentrations in samples collected ten minutes after completion of theaerosol dose. This study administered TOBI® tobramycin solution forinhalation, PathoGenesis Corporation, Seattle, Wash. (now ChironCorporation, Emeryville, Calif.), containing 60 mg/mL tobramycin in 5 mLone quarter (1/4) normal saline, using the Pari® LC jet nebulizer, PariRespiratory Equipment, Inc., Richmond, Va. This delivery system wasshown to deliver a mean peak sputum tobramycin concentration of 678.8μg/g (s.d. 661.0 μg/g), and a median peak sputum concentration of 433.0μg/g. Only 13% of patients had sputum levels ≦128 μg/g; 87% of patientsachieved sputum levels of ≧128 μg/g (Eisenberg, J. et al., “A Comparisonof Peak Sputum Tobramycin Concentration in Patients With Cystic FibrosisUsing Jet and Ultrasonic Nebulizer Systems. Aerosolized Tobramycin StudyGroup,” Chest 111(4):955-962 (1997)). Recently, the Pari® LC jetnebulizer has been modified with the addition of one-way flow valves,and renamed the Pari® LC PLUS. The one-way valves in the Pari® LC PLUShave been described as permitting the delivery of more drug than thePari® LC jet nebulizer, while decreasing the potential for accidentalspillage and allowing for the use of an expiratory filter. Experiencehas shown that mean peak sputum tobramycin concentrations achieved usingthe Pari LC PLUS jet nebulizer are significantly higher than those usingthe Pari® LC jet nebulizer as described in Eisenberg et al. (1997),supra.

[0009] Two placebo-controlled, multicenter, randomized, double blindclinical trials of intermittent administration of inhaled tobramycin incystic fibrosis patients with P. aeruginosa infection were reported inRamsey, B. W. et al., “Intermittent Administration of Inhaled Tobramycinin Patients with Cystic Fibrosis. Cystic Fibrosis Inhaled TobramycinStudy Group.” N. Engl. J. Med. 340(1):23-30 (1999). In these studies,five hundred twenty subjects were randomized to receive either 300 mginhaled tobramycin or placebo twice daily for 28 days followed by 28days off study drug. Subjects continued on treatment or placebo forthree “on-off” cycles for a total of 24 weeks. Efficacy variablesincluded sputum P. aeruginosa density. Tobramycin-treated patients hadan average 0.8 log₁₀ decrease in P. aeruginosa density from Week 0 toWeek 20, compared with a 0.3 log₁₀ increase in placebo-treated patients(P<0.001). Tobramycin-treated patients had an average 1.9 log₁₀ decreasein P. aeruginosa density from Week 0 to Week 4, compared with no changein placebo-treated patients (P<0.001).

[0010] A preservative-free, stable, and convenient formulation oftobramycin (TOBI® tobramycin solution for inhalation; 60 mg/mLtobramycin in 5 mL of 1/4 normal saline) for administration via jetnebulizer was developed by PathoGenesis Corporation, Seattle, Wash. (nowChiron Corporation, Emeryville, Calif.). The combination of a 5 mL BIDTOBI dose (300 mg tobramycin) and the PARI LC PLUS/PulmoAide compressordelivery system was approved under NDA 50-753, December 1997, for themanagement of P. aeruginosa in CF patients, and remains the industrystandard for this purpose. The aerosol administration of a 5 ml dose ofa formulation containing 300 mg of tobramycin in quarter normal salinefor the suppression of P. aeruginosa in the endobronchial space of apatient is disclosed in U.S. Pat. No. 5,508,269, the disclosure of whichis incorporated herein in its entirety by this reference.

[0011] Although the current conventional delivery systems have beenshown to be clinically efficacious, they typically suffer fromrelatively low efficiency levels in delivering antibiotic solutions tothe endobronchial space of a patient, thereby wasting a substantialportion of the nebulized antibiotic formulations and substantiallyincreasing drug delivery costs. The low efficiency of currentconventional delivery systems requires patients to devote relativelylong time periods to receive an effective dose of the nebulizedantibiotic formulations, which can lead to decreased patient compliance.Accordingly, there is a need for new and improved methods and devicesfor the delivery of aminoglycoside antibiotic compounds to a patient byinhalation to reduce administration costs, increase patient complianceand enhance overall effectiveness of the inhalation therapy.

SUMMARY OF THE INVENTION

[0012] It has now been discovered that patients suffering from anendobronchial infection can be effectively and efficiently treated byadministering to the patient for inhalation a dose of less than about4.0 ml of a nebulized aerosol formulation comprising from about 60 toabout 200 mg/ml of an aminoglycoside antibiotic, such as tobramycin, ina physiologically acceptable carrier in a time period of less than about10 minutes, more preferably less than about 8 minutes, and even morepreferably less than about 6 minutes. In other aspects, the administereddose may be less than about 3.75 ml or 3.5 ml or less, and theaminoglycoside antibiotic formulation may comprise from about 80 toabout 180 mg/ml of aminoglycoside antibiotic or more preferably fromabout 90 to about 150 mg/ml of aminoglycoside antibiotic.

[0013] In other aspects, the present invention provides unit doseformulations and devices adapted for use in connection with a highefficiency inhalation system, the unit dose device comprising acontainer designed to hold and store the relatively small volumes of theaminoglycoside antibiotic formulations of the invention, and to deliverthe formulations to an inhalation device for delivery to a patient inaerosol form. In one aspect, a unit dose device of the inventioncomprises a sealed container, such as an ampoule, containing less thanabout 4.0 ml of an aminoglycoside antibiotic formulation comprising fromabout 60 to about 200 mg/ml of an aminoglycoside antibiotic in aphysiologically acceptable carrier. The sealed container is preferablyadapted to deliver the aminoglycoside antibiotic formulation to a highefficiency inhalation device for aerosolization and inhalation by apatient. In other aspects, the container of the unit dose device maycontain less than about 3.75 ml, or 3.5 ml or less, of theaminoglycoside antibiotic formulation, and the aminoglycoside antibioticformulation may comprise from about 80 to about 180 mg/ml, or from about90 to about 120 mg/ml, of aminoglycoside antibiotic.

[0014] In yet other aspects, the present invention relates to a systemfor delivering an aminoglycoside antibiotic formulation to a patient inneed of such treatment, comprising a unit dose device as described indetail above, comprising a container containing less than about 4.0 mlof an aminoglycoside antibiotic formulation comprising from about 60 toabout 200 mg/ml of an aminoglycoside antibiotic in a physiologicallyacceptable carrier, and means for delivering the aminoglycosideantibiotic formulation from the unit dose device for inhalation by thepatient in aerosolized form in less that 10 about minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The foregoing aspects and many of the attendant advantages ofthis invention will become more readily appreciated as the same becomebetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

[0016]FIG. 1 is a graphical representation illustrating the meanrelative changes in FEV₁ % predicted from before to 30 minutes afterdosing with 300 mg tobramycin with a PARI LC PLUS jetnebulizer/PulmoAide compressor delivery system, or with 30, 60, or 90 mgtobramycin with an Aerodose breath actuated nebulizer, as described inExample 1;

[0017]FIG. 2 is a graphical representation showing sputum tobramycinconcentrations by time from dosing by the tobramycin formulations ofFIG. 1, as described in Example 1;

[0018]FIG. 3 is a graphical representation showing sputum maximum plasmaconcentrations (C_(max)) following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

[0019]FIG. 4 is a graphical representation showing sputum area under theplasma concentration time profile (AUC₀₋₈) following dosing by thetobramycin formulations of FIG. 1, as described in Example 1;

[0020]FIG. 5 is a graphical representation showing serum tobramycinconcentrations by time following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

[0021]FIG. 6 is a graphical representation showing serum maximum plasmaconcentrations (C_(max)) following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

[0022]FIG. 7 is a graphical representation showing serum area under theplasma concentration time profile (AUC₀₋₈) following dosing by thetobramycin formulations of FIG. 1, as described in Example 1;

[0023]FIG. 8 is a graphical representation showing the mean recovery oftobramycin from urine 0-8,8-24 and 0-24 hours post dosing with theformulations of FIG. 1, as described in Example 1; and

[0024]FIG. 9 is a graphical representation showing the mean nebulizationtime in minutes for dosing with the formulations of FIG. 1, as describedin Example 1.

[0025]FIG. 10 is a graphical representation showing the averageserum-time profiles of tobramycin after administration of 300 mgtobramycin (TOBI) and 420 mg tobramycin solution for inhalation (TSI),as described in Example 3.

[0026]FIG. 11 is a graphical representation showing the averagesputum-time profiles of tobramycin after administration of 300 mgtobramycin (TOBI) and 420 mg tobramycin solution for inhalation (TSI),as described in Example 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0027] In accordance with the present invention, methods are providedfor the treatment of a patient in need of treatment, such as a patientsuffering from an endobronchial P. aeruginosa infection, comprisingadministering to the patient for inhalation a relatively small volume ofan aminoglycoside antibiotic formulation over a relatively short periodof time. This aspect of the invention is particularly suitable forformulation of concentrated aminoglycosides, such as tobramycin, foraerosolization by small volume, breath actuated, high output rate andhigh efficiency inhalers to produce a aminoglycoside aerosol particlesize between 1 and 5 μm desirable for efficacious delivery of theaminoglycoside into the endobronchial space to treat susceptiblemicrobial infections, such as Pseudomonas aeruginosa infections. Theformulations preferably contains minimal yet efficacious amount ofaminoglycoside formulated in smallest practical volume of aphysiologically acceptable solution, for example an aqueous solutionhaving a salinity adjusted to permit generation of aminoglycosideaerosol particles that are well-tolerated by patients but preventing thedevelopment of secondary undesirable side effects such as bronchospasmand cough. By the more efficient administration of the aminoglycosideformulation provided by the present invention, substantially smallervolumes of aminoglycoside than the conventional administration regimeare administered in substantially shorter periods of time, therebyreducing the costs of administration and drug waste, and significantlyenhancing the likelihood of patient compliance.

[0028] Thus, in accordance with one aspect of the present invention,methods are provided for the treatment of a patient in need oftreatment, such as a patient suffering from an endobronchial P.aeruginosa infection, comprising administering to the patient forinhalation a dose of less than about 4.0 ml of a nebulized aerosolformulation comprising from about 60 to about 200 mg/ml of anaminoglycoside antibiotic in a time period of less than about 10minutes. In other aspects, the dose of the aerosol formulation isadministered to the patient in less than about 8 minutes. In yet otheraspects, the dose of the aerosol formulation is administered to thepatient in less than about 6 minutes.

[0029] The aerosol formulations administered in the practice of theinvention may comprise from about 60 to about 200 mg/ml ofaminoglycoside antibiotic. In other aspects of the invention, theaerosol formulations administered in the practice of the invention maycomprise from about 80 to about 180 mg/ml of aminoglycoside antibiotic.In yet other aspects of the invention, the aerosol formulationsadministered in the practice of the invention may comprise from about 90to about 150 mg/ml of aminoglycoside antibiotic.

[0030] In the practice of the methods of the invention, substantiallysmaller volumes of aerosol formulation are administered to the patient,as compared with the conventional administration processes. Thus, in oneaspect a dose of less than about 4.0 ml of a nebulized aerosolformulation is administered to the patient. In another aspect, a dose ofless than about 3.75 ml of a nebulized aerosol formulation isadministered to the patient. In yet another aspect, a dose of 3.5 ml orless of a nebulized aerosol formulation is administered to the patient.

[0031] In yet other aspects, the present invention relates to a systemfor delivering an aminoglycoside antibiotic formulation to a patient inneed of such treatment, comprising a unit dose device as described indetail herein, comprising a container containing less than about 4.0 mlof an aminoglycoside antibiotic formulation comprising from about 60 toabout 200 mg/ml of an aminoglycoside antibiotic in a physiologicallyacceptable carrier, and means for delivering the aminoglycosideantibiotic formulation from the unit dose device for inhalation by thepatient in aerosolized form in less that 10 about minutes.

[0032] In order to deliver the relatively small volumes of therelatively high concentration aminoglycoside antibiotic formulations tothe patient for inhalation in the relatively short dosing periods of theinvention, the antibiotic formulations are preferably administered withthe use of an inhalation device having a relatively high rate of aerosoloutput. Useful devices may also exhibit high emitted dose efficiency(i.e., low residual volume in the device). In order to increase theoverall efficiency of the system, emission may additionally be limitedto periods of actual inhalation by the patient (i.e., breath actuated).Thus, while conventional air-jet nebulizers exhibit a rate of aerosoloutput on the order of 3 μl/sec, inhalation devices useful for use inthe practice of the present invention will typically exhibit a rate ofaerosol output of not less that about 4 μl/sec. In some cases,inhalation devices useful for use in the practice of the presentinvention will exhibit a rate of aerosol output of not less than about 5μl/sec or even not less than about 8 μl/sec. In addition, whileconventional air-jet nebulizers have a relatively low emitted doseefficiency and typically release about 55% (or less) of the nominal doseas aerosol, inhalation devices useful for use in the practice of thepresent invention may release at least about 75%, more preferably atleast about 80% and most preferably at least about 85% of the loadeddose as aerosol for inhalation by the patient. In other aspects,conventional air-jet nebulizers typically continually releaseaerosolized drug throughout the delivery period, without regard towhether the patient is inhaling, exhaling or in a static portion of thebreathing cycle, thereby wasting a substantial portion of the loadeddrug dose. In some embodiments, inhalation devices for use in thepresent invention will be breath actuated, and restricted to delivery ofaerosolized particles of the aminoglycoside formulation to the period ofactual inhalation by the patient. One representative inhalation devicemeeting the above criteria and suitable for use in the practice of theinvention is the Aerodose™ inhaler, available from Aerogen, Inc.,Sunnyvale, Calif. The Aerodose inhaler generates an aerosol using aporous membrane driven by a piezoelectric oscillator. Aerosol deliveryis breath actuated, and restricted to the inhalation phase of the breathcycle, i.e., aerosolization does not occur during the exhalation phaseof the breath cycle. The airflow path design allows normal inhale-exhalebreathing, compared to breath-hold inhalers. Additionally, the Aerodoseinhaler is a hand-held, self-contained, and easily transported inhaler.Although piezoelectric oscillator aerosol generators, such as theAerodose™ inhaler, represent one embodiment for use in the practice ofthe invention, other inhaler or nebulizer devices may be employed thatmeet the above performance criteria and are capable of delivering thesmall dosage volumes of the invention with a relative high effectivedeposition rate in a comparatively short period of time. In otherembodiments of the invention devices useful for delivering theconcentrated aminoglycoside formulations of the invention includeconventional air-jet nebulizers coupled with a compressor capable ofhigher than conventional output pressures. Enhanced compressor outputpressures useful in the practice of the invention will be readilydeterminable to those skilled in the art in view of the disclosurecontained herein. As one representative example, the PARI LC PLUS™ jetnebulizer, PARI GmbH, Starnberg, Germany, driven by a InvacareMOBILAIRE™ compressor, Invacare Corporation, Elyria, Ohio, set for anoutput pressure of about 35 psi has been found to be capable ofdelivering 3.5 ml of the concentrated aerosolized aminoglycosideformulations of the invention (such as tobramycin) in 10 minutes orless, as is hereinafter described in detail in Example 3.

[0033] Aminoglycoside antibiotics useful in the practice of theinvention include, for example, gentamicin, amikacin, kanamycin,streptomycin, neomycin, netilmicin and tobramycin. A presentlyparticularly preferred aminoglycoside antibiotic for this purpose istobramycin. Formulations according to the invention typically containfrom about 60 to about 200 mg, more preferably from about 80 to about180, and most preferably from about 90 to about 120 mg of aminoglycosideper ml of solution. The aminoglycoside antibiotic of the invention maybe incorporated into sterile water or physiologically acceptablesolution. Other components may be included in the formulation, asdesired. In order to facilitate administration and compatibility withthe endobronchial space, the aminoglycoside antibiotic of the inventionis preferably formulated in a diluted physiological saline solution,such as in one quarter strength of normal saline, having a salinityadjusted to permit generation of tobramycin aerosol well-tolerated bypatients but to prevent the development of secondary undesirable sideeffects such as bronchospasm and cough. Typically, about 90 to about 120mg of aminoglycoside antibiotic is dissolved in 1 ml solution of adiluted, typically quarter normal saline containing about 0.225% NaCl.Quarter normal saline, that is 0.225% of sodium chloride, is a presentlypreferred vehicle for delivery of aminoglycoside into endobronchialspace.

[0034] By way of illustration, high concentrations of tobramycinadministered to the lungs by aerosolization result in maximization ofsputum levels of tobramycin and in minimization of tobramycin serumlevels. Thus, administration of tobramycin by aerosolization has theadvantage of reducing systemic toxicity while providing efficaciousconcentrations of tobramycin in the sputum. The bronchial barrierrestricts the movement of aerosolized tobramycin and prevents it fromreaching high systemic levels.

[0035] In other aspects of the present invention, unit dose formulationsand devices are provided for administration of an aminoglycosideantibiotic formulation to a patient with an inhaler, in accordance withthe methods of the invention as described supra. Preferred unit dosedevices comprise a container designed to hold and store the relativelysmall volumes of the aminoglycoside antibiotic formulations of theinvention, and to deliver the formulations to an inhalation device fordelivery to a patient in aerosol form. In one aspect, unit dosecontainers of the invention comprise a plastic ampoule filled with anaminoglycoside antibiotic formulation of the invention, and sealed understerile conditions. Preferably, the unit dose ampoule is provided with atwist-off tab or other easy opening device for opening of the ampouleand delivery of the aminoglycoside antibiotic formulation to theinhalation device. Ampoules for containing drug formulations are wellknown to those skilled in the art (see, for example, U.S. Pat. Nos.5,409,125, 5,379,898, 5,213,860, 5,046,627, 4,995,519, 4,979,630,4,951,822, 4,502,616 and 3,993,223, the disclosures of which areincorporated herein by this reference). The unit dose containers of theinvention may be designed to be inserted directly into an inhalationdevice of the invention for delivery of the contained aminoglycosideantibiotic formulation to the inhalation device and ultimately to thepatient.

[0036] In accordance with this aspect of the invention, a unit dosedevice is provided comprising a sealed container containing less thanabout 4.0 ml of an aminoglycoside antibiotic formulation comprising fromabout 60 to about 200 mg/ml of an aminoglycoside antibiotic in aphysiologically acceptable carrier, the sealed container being adaptedto deliver the aminoglycoside antibiotic formulation to an inhalationdevice for aerosolization. Suitable aminoglycoside antibiotics for usein connection with this aspect of the invention include thoseaminoglycoside antibiotics described in detail, supra. In a presentlypreferred embodiment, the aminoglycoside antibiotic employed in the unitdose devices of the invention is tobramycin. In other aspects, the unitdose devices of the invention contain less than about 3.75 ml of theaminoglycoside solution. In other aspects, the unit dose devices of theinvention contain 3.5 ml or less of the aminoglycoside solution.

[0037] In other aspects of the invention, the unit dose devices of theinvention may contain an aminoglycoside antibiotic formulationcomprising from about 80 to about 180 mg/ml of aminoglycosideantibiotic. In yet other aspects of the invention, the unit dose devicesof the invention may contain an aminoglycoside antibiotic formulationcomprising from about 90 to about 150 mg/ml of aminoglycosideantibiotic.

[0038] In preferred unit dose formulations of the invention, thephysiologically acceptable carrier may comprise a physiological salinesolution, such as a solution of one quarter strength of normal saline,having a salinity adjusted to permit generation of a tobramycin aerosolthat is well-tolerated by patients, but that prevents the development ofsecondary undesirable side effects such as bronchospasm and cough.

[0039] These and other aspects of the inventive concepts may be betterunderstood in connection with the following non-limiting examples.

EXAMPLES Example 1 In Vivo Study 1

[0040] A comparison was made of the safety, pharmacokinetics, aerosoldelivery characteristics, and nebulization time of the conventional doseand inhalation delivery system (5 mL ampoule containing 300 mgtobramycin and 11.25 mg sodium chloride in sterile water for injection(TOBI® tobramycin solution for inhalation, Chiron Corporation, Seattle,Wash.), pH 6.0; administered with a PARI LC PLUS™ jet nebulizer with aPulmoAide compressor) with 3 doses of TOBI (30 mg tobramycin in 0.5 mLsolution, 60 mg in 1.0 mL, and 90 mg in 1.5 mL) using a TM AeroDose™inhaler device.

[0041] The study was designed as an open label, randomized, multicenter,single dose, unbalanced, four treatment, three period crossover trial.Each patient was to receive three single doses of aerosolizedantibiotic: the active drug control treatment during one treatmentperiod and two of three experimental treatments during two additionaltreatment periods. Single dose administration during the three treatmentperiods was to occur at one-week intervals.

[0042] In accordance with the study design, forty eight eligible maleand female patients 12 years of age or older with a confirmed diagnosisof cystic fibrosis were to be enrolled in the study and randomlyassigned to one of 12 treatment sequences of three treatments each (oneactive control and two experimental treatments) with the constraint thatthe active control treatment was to be administered in either the firstor the second of the three treatment periods. Experimental treatmentswere administered during all three treatment periods. Each patientinhaled a single dose of aerosolized control and two of threeexperimental treatments in accordance with the present invention asfollows.

[0043] control delivery treatment (PARI LC PLUS jet nebulizer+PulmoAidecompressor):

[0044] TOBI 300 mg in 5 mL solution.

[0045] experimental delivery treatments (AeroDose™ inhaler breathactuated nebulizer):

[0046] TOBI 30 mg in 0.5 mL solution;

[0047] TOBI 60 mg in 1.0 mL solution;

[0048] TOBI 90 mg in 1.5 mL solution.

[0049] The duration of study participation for each patient was to beapproximately five weeks including a brief (2 days to one week)screening period, three one-week treatment periods, and a one-weektelephone follow-up period.

[0050] Control and Experimental Treatments

[0051] Each patient was to self-administer under research staffsupervision a total of three single doses of aerosolized tobramycinduring the study, one dose per crossover treatment period. Patients wereto receive a single dose of the control delivery treatment during period1 or period 2 of the three treatment periods. In addition, each patientwas to receive single doses of two of the three experimental deliverytreatments during the remaining two treatment periods. Control andexperimental delivery treatments were specified as follows.

[0052] Control Delivery Treatment:

[0053] PARI LC PLUS jet nebulizer with PulmoAide compressor:preservative free tobramycin 60 mg/mL (excipient 5 mL of 1/4 normalsaline adjusted to a pH of 6.0±0.5); 300 mg in 5 mL.

[0054] Experimental Delivery Treatments:

[0055] Aerodose with a 3-4 μm mass medium diameter (MMD) aerosolparticle size: preservative free tobramycin 60 mg/mL (excipient 0.5 mLof 1/4 normal saline adjusted to a pH of 6.0±0.5); 30 mg in 0.5 mL;

[0056] Aerodose with a 3-4 μm MMD: preservative free tobramycin 60 mg/mL(excipient 1.0 mL of ¼ normal saline adjusted to a pH of 6.0±0.5); 60 mgin 1.0 mL;

[0057] Aerodose with a 3-4 μm MMD: preservative free tobramycin 60 mg/mL(excipient 1.5 mL of ¼ normal saline adjusted to a pH of 6.0±0.5); 90 mgin 1.5 mL.

[0058] Patients were placed upright in a sitting or standing position topromote normal breathing and were instructed to place the nose clipsover the nostrils and to breath normally through the mouth until therewas no longer any mist produced by the nebulizer. Aerosol delivery wasanticipated to take 15 minutes to complete.

[0059] A pharmacist or coordinator prepared the 30 mg dose of TOBI bydrawing 0.5 mL of the 60 mg/mL TOBI formulation into a one-mL syringe.Each syringe was labeled with the patient identification number. Studydrug was dispensed into the medication reservoir as indicated in theAerodose directions for use. TOBI 60 mg and 90 mg doses were similarlyprepared by drawing two and three 0.5 mL aliquots, respectively, fromthe TOBI ampoule into two and three one-mL syringes.

[0060] Aerosol Delivery Systems

[0061] The control delivery system (PARI LC PLUS jet nebulizer) was usedonce per patient during the study for administration of TOBI 300 mg(control treatment). The experimental delivery system (Aerodose inhaler)was used to deliver only one dose of study treatments.

[0062] The control nebulizer, the PARI LC PLUS jet nebulizer withDeVilbiss PulmoAide compressor, generates aerosol by air-jet shear. Adetailed comparison of experimental and control devices is provided inTable 1. TABLE 1 DEVICE COMPARISON PARI LC PLUS Nebulizer Aerodose andDe Vilbiss Device Characteristic Nebulizer PulmoAide Compressor Aerosolgenerating Piezoelectric Air-jet shear principle vibration Aerosolcharacteristics with TOBI Mass median diameter 4.0 pm 4.8 μm (MMD)Output rate 8.0 μL/sec 3.6 μL/sec Emitted dose 85% 57% Dose actuationBreath-actuated by On/off switch; when on, user inhalation medicationaerosolized continuously Control of aerosol Breath actuated. AnContinuous aerosol output generation airflow sensor during bothinhalation and system is used to exhalation limit aerosol generation toinhalation User indicator lights Green LED flashing None for “deviceready” and solid for “aerosolization” Red LED for “low battery” Physicalcharacteristics 3.3″ × 2.6″ × 1.1″ 7.5″ × 7.5″ × 3.0″ (nebulizer) Size10.1″ × 10.5″ × 6.5″ (compressor) Weight 140 gm 68 gm (nebulizer) 3,200gm (compressor) Power source Four AAA alkaline 115 VAC, 60 Hz batteriesPower consumption 2.5 watts 90 watts (max.) Where used Fully portableRestricted to power outlets supplying 115 VAC, 60 Hz

[0063] Selection of Doses in the Study

[0064] Commercial TOBI 60 mg/mL in 5 mL solution administered by PARI LCPLUS jet nebulizer and powered by the PulmoAide compressor was theactive drug control delivery system against which potential improvementsin aerosol delivery technology by the Aerodose breath actuated nebulizerwere compared in this example.

[0065] The selection of doses of experimental treatments (TOBI 30 mg in0.5 mL solution, 60 mg in 1.0 mL, and 90 mg in 1.5 mL) was based onempirical data on the comparative predicted efficiency of the Aerodoseinhaler relative to the PARI LC PLUS nebulizer. The selection of doseswas also based on the assumption that TOBI delivered via the PARI LCPLUS jet nebulizer leads to the systemic absorption of approximately11.7% of the administered dose {Pitlick, Nardella, et al., 1999}.Furthermore, the mean and standard deviation of the serum concentrationone hour after inhalation was 1.0 μg/mL ±0.58, suggesting a wide rangeof deposition (Table 5.2 C, Clinical Pharmacology, PathoGenesis NDA,#50,753). Due to design features of the Aerodose inhaler, it wasestimated that between 50-70% of the drug would be delivered to thelung. This assumption is based on the predicted efficiency of anebulized dose.

[0066] Patients were randomized to treatment sequence groups, andpredose procedures were completed including a physical examination (onlyif abnormal during screening), recheck of inclusion and exclusioncriteria, interim history review, spirometry, clinical evaluation, andblood and urine specimens for laboratory tests (only if abnormal duringscreening). A bronchodilator was to be administered before dosing ifregularly used by the patient. Spirometry was completed 15-60 minutesafter the bronchodilator, if applicable.

[0067] Patients received a single dose of study treatments during eachof three treatment periods separated by an interval of 7 days betweentreatments. At the time of single dose administration during eachperiod, patients were instructed to sit upright and use nose clipsduring aerosol dose administration.

[0068] Patients remained at the clinic through completion of 8-hour posttreatment procedures (nebulization time, spirometry, and sputum, serumand urine specimens for tobramycin determinations). Patients were thendischarged from the clinic and were expected to collect and return their8-24 hour urine collection at the next visit, no later than 7 days aftertheir previous visit. Patients were to refrigerate urine collections atall times except during transport.

[0069] Safety Variables

[0070] Safety was assessed by monitoring the incidence of bronchospasmand by the quantitative change in pulmonary function (measured as changein FEV₁ % predicted), the incidence of treatment emergent adverseevents, and the incidence of unusually high serum tobramycin results (>4μg/mL), the significance of clinical laboratory test results, and thesignificance of change in clinical evaluation results.

[0071] Bronchospasm (Airway Reactivity)

[0072] One objective of the study was to compare the rate of occurrenceof bronchospasm (airway reactivity) between control and experimentaldelivery systems. Bronchospasm was measured by the change in forcedexpiratory volume in 1 second [FEV₁ (liters)] from before dosing to 30minutes after dosing during periods 1, 2, and 3. The number and percentof patients who experienced predose to postdose decreases in FEV₁(liters) that were ≧10% and those that were ≧20% were determined toassess the comparative incidence of bronchospasm among control andexperimental treatments. Decreases in FEV₁ (liters) that were ≧20% wereconsidered clinically significant for the purposes of the study.Additionally, an acute decrease in FEV₁ (liters)≧30% from before toafter treatment was considered a symptom of respiratory distress. Inthis event, continuation of the patient in the study was at thediscretion of the investigator.

[0073] Norms have been developed for FEV₁. These norms are commonly usedin studies of pulmonary patients. This study employed the Knudsonequations that use age, gender, and height to predict a patient's FEV₁values as if the patient was free of pulmonary function disease. Theactual FEV₁ value is divided by the normative value, and the resultingratio is multiplied by 100 to produce a measure that representspercentage of predicted normal function, commonly called percentpredicted. The transformation is:

FEV ₁ % predicted=(FEV _(1 actual value) /FEV _(1 normative value))×100

[0074] Relative change in FEV₁ % predicted is defined as the percentchange from predose to 30 minutes postdose in FEV₁ % predicted and iscalculated as:

[0075] relative change in

FEV ₁ % predicted=[(FEV _(1 (% predicted at 30 minutes postdose)) −FEV_(1 (% predicted at predose)))/FEV _(1 (% predicted at predose))]×100

[0076] Clinical Laboratory Tests

[0077] Serum creatinine, blood urea nitrogen (BUN), and dipstick urineprotein results were obtained from specimens drawn during screening andbefore dosing during treatment period 3. Urine dipstick testing wasalways performed on fresh specimens. Serum and urine specimens thatneeded to be retained at the site (e.g., those drawn after shippingpick-up hours or on Friday or Saturday) were frozen until shipment atthe next earliest shipping time. Specimens were covered with dry ice forshipping.

[0078] All out of range laboratory results were evaluated for clinicalsignificance and drug relationship by the investigator using thefollowing classification scheme:

[0079] clinically insignificant;

[0080] possible study medication relationship;

[0081] probable study medication relationship;

[0082] unrelated to study medication, related to concurrent illness;

[0083] unrelated to study medication, related to other concurrentmedication;

[0084] other (investigator commentary).

[0085] Aerosol Delivery Variables

[0086] Evaluation of the aerosol delivery characteristics of theAerodose breath actuated nebulizer, compared to characteristics of theFDA-approved PARI LC PLUS jet nebulizer with PulmoAide compressor, wasbased on determination of sputum, urine, and serum tobramycinconcentrations, calculation of certain sputum and serum pharmacokineticparameters, and measurement of nebulization time.

[0087] Sputum Tobramycin Concentrations

[0088] Before study treatments were administered, patients expectoratedsputum produced from a deep cough into an individual specimen container.Immediately after treatment, patients rinsed their mouths three timeswith 30 mL of normal saline, gargled for 5-10 seconds, and expectoratedthe rinse.

[0089] Post treatment sputum specimens were collected following thenormal saline gargle at 10 minutes and at 1, 2, 4, and 8 hours aftercompletion of the aerosol drug administration for determination ofsputum tobramycin concentrations. Sputum specimens were judged to beacceptable if collected within ±2 minutes of the scheduled 10-minuteposttreatment collection time and within ±10 minutes of the scheduled1-, 2-, 4-, and 8-hour collection times. After collection, specimenswere immediately frozen for later determination of tobramycinconcentrations in sputum. A minimum of 1 gram of sputum was required foranalysis. Tobramycin concentrations in sputum (sputum LOQ 20.0 μg/gm)were measured by using HPLC.

[0090] Serum Tobramycin Concentrations

[0091] Whole blood was drawn by venipuncture, an indwellingheparin/saline lock, or a permanent venous access port at 10 minutes andat 1, 2, 4, and 8 hours after completion of dosing. Blood specimens werejudged to be acceptable if collected within ±2 minutes of the scheduled10-minute posttreatment collection time and within ±10 minutes of thescheduled 1-, 2-, 4-, and 8-hour collection times. Blood specimens wereallowed to clot for 30 minutes and were then centrifuged at 1500× g for10 minutes until clot and serum separated. Serum samples (3 mL) werepipetted into plastic vials and frozen immediately for laterdetermination of serum tobramycin concentrations.

[0092] Tobramycin concentrations in serum were measured by AbbottTDxFLx® assay (Abbott Laboratories, Abbott Park, Ill.) [serum lowerlimit of quantitation (LOQ)=0.18 μg/mL].

[0093] Urine Tobramycin Recovery

[0094] Urine specimens were collected and combined in a 24-hourcollection container during the 12 hours before treatment (-12-0 hourperiod) and during 0-8 hour and 8-24 hour collection periods aftertreatment according to instructions provided in the Study Manual. Totalurine volume for the collection period was recorded, and a 10 mL aliquotfrom each urine collection was retained and frozen for later analysis ofurine tobramycin concentration.

[0095] The recovery of tobramycin in urine (in milligrams) during 0-8hour and 8-24 hour collection periods was calculated as follows.

urine tobramycin recovery (μg)=urine volume (mL)·urine tobramycinconcentration (μg/mL)

[0096] Urine tobramycin recovery was normalized for each collectionperiod according to TOBI dose as follows.

dose-normalized urine tobramycin recovery (μg/mg)=[urine tobramycinrecovery (μg)÷TOBI dose (mg)]

[0097] The percent of the TOBI dose excreted in urine in the 24-hourperiod following treatment was calculated as follows.

% tobramycin excreted in urine=[(urinary recovery in μg÷1000 μg/mg)÷TOBIdose in mg]·100%.

[0098] If either the urine volume or the urine tobramycin concentrationfor a collection interval was missing, then the urine tobramycinrecovery was not calculable for that interval. If calculated urinetobramycin recovery was missing for either the 0-8 hour or the 8-24 hourcollection interval, then the 0-24 hour urine tobramycin recovery wasnot calculated. Missing urine tobramycin recovery values were notreplaced by estimated values for analysis purposes.

[0099] Tobramycin concentrations in urine were measured by AbbottTDxFLx® assay [urine lower limit of quantitation (LOQ)=1.0 μg/mL].

[0100] Pharmacokinetic Parameters

[0101] The maximum tobramycin concentrations (C_(max)) in sputum andserum during the 8-hour posttreatment sampling period were identifiedfor each patient during each treatment period, and the time at whichC_(max) was observed (T_(max)) was recorded.

[0102] Area under the concentration-time curve through 8 hours postdose(AUCO₀₋₈) was calculated from sputum and serum tobramycin concentrationsusing the linear trapezoidal method. Nebulization time (excluding timefor refilling) was added to the time between predose and 10 minutespostdose for AUC₀₋₈ calculations.

[0103] Area under the concentration-time curve extrapolated to infinity(AUC_(0-∞)) was calculated for sputum and serum as follows.

AUC _(0-∞) =AUC _(0-last) +C _((last)) ÷k _(el)

[0104] where: AUC_(0-last) is area under the curve from predose throughthe last non-BQL time

[0105] C_((last)) is the last non-BQL posttreatment concentration resultk_(el) is the elimination rate constant (terminal phase slope)

[0106] and k_(el)=log 2÷T_(1/2)

[0107] where T_(1/2) is the elimination half-life for the patient.

[0108] Relative systemic bioavailability was calculated based on serumAUC₀₋₈ values for control (TOBI 300 mg delivered by PARI LC PLUSnebulizer) and experimental (TOBI 30 mg, 60 mg, and 90 mg delivered byAerodose inhaler) groups as follows.

relative bioavailability (%)=experimental group serum AUC ₀₋₈÷controlgroup serum AUC ₀₋₈

[0109] Missing tobramycin concentrations and those reported as zero orbelow quantifiable limits (BQL) were not to be replaced with anyestimated value. C_(max) and AUC₀₋₈ were always determinable except inthe event that all posttreatment tobramycin concentrations were missing,zero, or BQL. There was no missing sputum C_(max) and AUC₀₋₈ valuesamong the 49 patients who completed the study (refer to report section9.3.1 for details). Four completing patients had indeterminate serumC_(max) and AUC₀₋₈ values due to BQL serum results for eachposttreatment sampling time (refer to report section 9.4.1 for details).

[0110] Nebulization Time

[0111] The timing (duration) of nebulization began with the patient'sfirst tidal breath after the device was in place and continued until thedevice aerosolized no more TOBI solution. Nebulization time did notinclude any interruptions or time needed TM for instillation of druginto the nebulizer between the repeat filling of the AeroDose™ inhaler.The length of any interruption in nebulization and the reason forinterruption were recorded.

[0112] Safety Analyses

[0113] Reductions in FEV₁ % predicted ≧10% and ≧20% were used asindicators of the occurrence of bronchospasm (airway reactivity).McNemar's test for paired comparisons (replacing theCochran-Mantel-Haenszel (CMH) test) was used for control vs.experimental treatment comparisons of the incidence of patients withpredose to 30-minute postdose decreases in FEV₁ % predicted that were≧10% and ≧20%. In addition, pairwise t-tests were used to compare meanrelative change in spirometry FEV₁ % predicted from predose to postdosebetween each experimental treatment and the control treatment. Allstatistical analyses were performed using two-sided tests conducted at a0.05 significance level (i.e., α=0.05). Since all statistical tests wereexploratory in nature, no adjustment of p-values was made for multipletesting. Changes from predose to postdose in vital signs, body weight,and the incidence of abnormal and/or clinically significant laboratoryand physical examination results were summarized and evaluateddescriptively.

[0114] Individual patient serum tobramycin results were monitored forunusually high values (≧4 μg/mL) that might potentially indicate theoccurrence of systemic toxicity.

[0115] Aerosol Delivery Analyses

[0116] The natural logarithms of AUC₀₋₈, AUC_(0-∞). and C_(max) based onsputum and serum tobramycin concentrations were to be statisticallyanalyzed using a mixed-effect repeated-measure analysis of variancemodel containing treatment, sequence, period, and carryover as fixedeffects and patient as a random effect. In the planned analysis ofvariance model, sequence and carryover (treatment by period interaction)effects were confounded. The actual model used for the analysis wastherefore modified by dropping the sequence term so that the assessmentof carryover (i.e., treatment by period interaction) could proceed. WhenAUC_(0-∞) values were calculated, large outlier values were noted, andthe analysis for this parameter was dropped.

[0117] Three hypotheses regarding whether the experimental deliverytreatment of 30 mg, 60 mg, or 90 mg TOBI was equivalent to the controldelivery treatment of 300 mg TOBI were to be tested in the model. Theexperimental treatment to control ratio for each of the log AUC andC_(max) parameters was estimated with 90 percent confidence intervals(CIs). Upper and lower limits for the CIs were then obtained by backtransformation (i.e., by exponentiating the log values of the upper andlower limits) to the original scale of the parameter. If the CIs for theratio of experimental and control treatments contained the value of 1.0,it was concluded that the treatments were not significantly different atthe α=0.1 for the 90% CIs.

[0118] If demographic or baseline characteristics showed importantapparent differences between the three experimental AeroDose™ groupscompared to all patients, then the discrepant factor and its interactionwith the delivery treatment factor were to be added to the mixed-effectmodel. Exploratory evaluations of age, gender, body weight, and baselinepulmonary function (FEV₁ percent predicted) demonstrated no importanteffects on pharmacokinetic results.

[0119] Disposition of Patients

[0120] A total of 56 patients were screened for the study by the nineinvestigators. Fifty-three patients met entrance criteria, were enrolledin the study, and were randomized to one of the 12 sequences oftreatment administration identified in the randomization code. A totalof 3 patients failed to meet entrance criteria and were not enrolled inthe study: 2 patients had screening FEV₁ % predicted results that werebelow the 40% criterion required for entry, and one patient exhibiteddisqualifying serum creatinine, BUN, and/or proteinuria.

[0121] Accrual of the 53 randomized patients at 9 sites was as follows:3 sites randomized 8 patients each, 2 sites randomized 7 patients, 3sites randomized 4 patients, and one site randomized 3 patients. Fiftytwo patients received at least one dose of study treatments, and onepatient was enrolled and randomized but withdrew from the study beforethe first study treatment due to increased productive cough with asignificant decline in forced expiratory volume (FEV) since screening(both events and associated hyperventilation were considered SAEs due tohospitalization of the patient: included in study database).

[0122] Of the 52 patients who received study treatments, 49 patientscompleted the study, and 3 patients withdrew after having received onedose of study treatment. Two of the withdrawn patients discontinued thestudy during the control treatment period (TOBI 300 mg administered byPARI LC PLUS nebulizer), and one patient withdrew during the TOBI 90 mgby AeroDose™ inhaler treatment period.

[0123] Baseline Characteristics

[0124] Enrolled patients had documented laboratory (sweat chloride≧60mEq/L by quantitative pilocarpine iontophoresis test (QPIT) and/orgenotype with 2 identifiable mutations) and clinical evidence consistentwith a diagnosis of cystic fibrosis. Patients met all inclusion andexclusion criteria except for one patient whose pulmonary functionentrance requirement (FEV₁≧40% of predicted based on gender, age, andheight) was waived (the patient's screening FEV₁ % predicted was39.87%). The average FEV₁ % predicted of all randomized patients was66.4% at screening with a range from approximately 40% to 116%.

[0125] Patients reported no known local or systemic hypersensitivity toaminoglycosides. Patients had taken no loop diuretics, no form ofaminoglycoside within 7 days before study treatments, and noinvestigational medications within 2 weeks before study treatments.

[0126] Female patients had a negative pregnancy test before studytreatments, and all patients had serum creatinine <2.0 mg/dL, BUN<40mg/dL, and <2+proteinuria at visit 1 screening, as required by theprotocol. Screening or repeat serum creatinine and BUN results werewithin the normal ranges for these tests before study treatments.Screening or repeat urine protein results were positive 1+in 3 patients,but this result did not preclude participation of these patients in thestudy.

[0127] No disqualifying medical history or physical examination findingswere noted at visit 1 screening. Screening and visit 1 predose vitalsigns were unremarkable for nearly all patients. One patient exhibitedlow systolic and diastolic blood pressures at (72/49 mmHg), but theseresults did not preclude participation of the patient in the study.

[0128] Safety Evaluation

[0129] Extent of Exposure

[0130] Forty-nine patients received all 3 single doses of studytreatments according to the randomization code, and 3 patients whowithdrew from the study received one dose of study treatment. These 52patients were included in the safety evaluation. Fifty-one of the 52patients received a single dose of TOBI 300 mg, and 34, 32, and 33 ofthe 52 patients received a single dose of TOBI 30 mg, 60 mg, and 90 mg,respectively. Three of the 49 completing patients had to stop treatmentdue to inhaler malfunction and subsequently repeated the treatmentperiod at a later date. As a result, these 3 patients received a partialdose of TOBI during the period in which the malfunction occurred (theamount of the partial dose was not recorded) and a full dose of TOBIduring the repeated period.

[0131] Pulmonary Function Results

[0132] Bronchospasm

[0133] In one aspect, the study compared the rate of occurrence ofbronchospasm (airway reactivity) between control and experimentaldelivery systems. The occurrence of bronchospasm was determinedquantitatively based on the percent change in FEV₁ (liters) from beforedosing to 30 minutes after dosing in each of the 3 treatment periods.For the purposes of the study, predose to postdose reductions in FEV₁(liters)≧10% and ≧20% were defined as bronchospasm; reductions in FEV₁(liters)≧20% were considered clinically significant.

[0134] Fifteen patients (9 male and 6 female) experienced 24 instancesof bronchospasm during the study. Two instances of clinicallysignificant bronchospasm were observed (decline in FEV₁ (liters)≧20%:patient 105-1034 after TOBI 300 mg and patient 102-1040 after TOBI 60mg). No statistically significant pairwise differences in the overallincidence of bronchospasm were noted between control and experimentaltreatments. No clear relationship appeared to exist between theincidence of bronchospasm and TOBI dose or delivery system (see Table 2below). TABLE 2 Incidence of Acute Bronchospasm by Treatment TOBI 30 mgTOBI 60 mg TOBI 90 mg Broncho- TOBI 300 mg Aerodose Aerodose Aerodosespasm PARI LC PLUS¹ inhaler² inhaler² inhaler² Parameter (N = 51) (N =34) (N = 32) (N = 33) FEV₁ 9 (17.6%) 5 (14.7%) 6 (18.8%) 4 (12.1%)Decrease ≧10% FEV₁ 1 (2.0%) 0 (0.0%) 1 (3.1%) 0 (0.0%) Decrease ≧20%

[0135] One patient 34 experienced clinically significant bronchospasm at30 minutes after completing the TOBI 300 mg dose during treatment period1 (visit 2). This 32-year old male patient's FEV₁ was 2.55 L beforedosing and 1.98 L (decline in FEV₁ (liters)≧20%) at 30 minutes afterdosing. He experienced moderate chest tightness that resolvedspontaneously. This patient also experienced a second episode ofbronchospasm 30 minutes after TOBI 60 mg during period 2. The FEV, was2.47 L before dosing and 2.14 L (decline in FEV₁ (liters)≧10% but<20%)at 30 minutes after dosing. No symptomatology was reported at the timeof this event. No prestudy aminoglycoside use was noted for thispatient.

[0136] One patient experienced one instance of clinically significantbronchospasm 30 minutes after TOBI 60 mg during period 3 (visit 4) ofthe crossover. This 36-year old male patient's FEV₁ was 2.26 L beforedosing and 1.75 L (decline in FEV, (liters)≧20%) at 30 minutes afterdosing (Archival Listing 3), but he reported no other symptomatology atthis time. No prestudy aminoglycoside use was noted for this patient.This episode of bronchospasm appeared due in part to anuncharacteristically high predose FEV₁ value. The 30-minuteposttreatment value was similar to that obtained during period 2 whenthe change in FEV₁ did not meet the definition of bronchospasm.

[0137] Among the 13 patients who experienced clinically non-significantbronchospasm, one patient experienced a decline in FEV₁ (liters)≧10%but<20% after all three study doses were administered, 6 patientsexperienced a decline in FEV₁ (liters)≧10% after two doses of studymedication, and 6 patients experienced a single instance ofbronchospasm. Table 3 below lists instances of bronchospasm by patient,treatment period, and TOBI dose. TABLE 3 Patient Dosing Regimen andAcute Bronchospasm Period 1 Period 2 Period 3 (Visit 2) (Visit 3) (Visit4) Site- TOBI Dose TOBI Dose TOBI Dose Patient ID/Gender ReceivedReceived Received 108-1048^(b)/Female 300^(c)  30^(c) 60109-1015^(b)/Male 300  30^(c) 60 107-1027/Male 300  30^(c) 90^(c)103-1038^(b)/Female 300^(c)  60 30 105-1034/Male 300^(d,e)  60^(c) 30107-1026/Female 300  60^(c) 90 102-1009^(b)/Female 300^(c)  90^(c) 30102-1040^(b)/Male 300  90 60^(d) 106-1050^(b)/Female  30^(c,e) 300 90102-1007^(b)/Male  60^(c) 300^(c) 30 104-1021/Male  60^(c) 300^(c) 30108-1044/Male  60 300^(c) 30^(c) 105-1047/Female  60 300^(c) 90106-1022^(b)/Male  90^(c,e) 300 30 106-1041^(b)/Male  90^(c,e) 300^(c)60^(c)

[0138] Three of the 15 patients with bronchospasm reportedtreatment-related symptoms at the same time. One patient 15 experiencedmoderate wheezing (coded as asthma) after TOBI 30 mg during period 2,one patient 4 experienced moderate chest tightness (coded as chest painas reported previously) after TOBI 300 mg during period 1, and onepatient 41 experienced increased cough after TOBI 60 mg during period 3.All events resolved either spontaneously (chest tightness), withtreatment (wheezing), or by holding and restarting therapy (increasedcough). None of the adverse events led to a serious outcome.

[0139] Four of the 15 patients with bronchospasm (and one patientwithout bronchospasm) reported “lung function decreased” (COSTART term)as an adverse event. In addition to the 4 patients with bronchospasmidentified in Table 3 above, one patient who experienced nobronchospasm, reported lung function decreased once after TOBI 60 mg andonce after TOBI 90 mg delivered by the AeroDose™ inhaler.

[0140] Initial instances of bronchospasm occurred more frequently duringperiod 1 than during periods 2 or 3 of the crossover. Nine of the 15patients first experienced bronchospasm during the first treatmentperiod (visit 2), five patients during the second treatment period, andone patient during the third treatment period.

[0141] Patients who routinely used a bronchodilator were permitted tocontinue to do so during the study. Bronchodilator doses were to beadministered 15 to 60 minutes prior to study treatments. Nine of the 15patients who experienced bronchospasm during the study used abronchodilator prior to administration of study treatment.

[0142] Relative Change in FEV₁ % Predicted

[0143] The magnitude of the relative change in FEV₁ % predicted wascalculated as a quantitative measure of the effect of TOBI treatments onpulmonary function during the study. There were no statisticallysignificant differences among the 4 treatments and no evidence of thepresence of period or carryover (treatment by period interaction)effects. Results of pairwise comparisons between control andexperimental treatments are summarized in Table 4. Since the overalltreatment difference was not statistically significant, the significantp-value for the TOBI 300 mg vs. TOBI 30 mg comparison in Table 4 below(p=0.019) should not be interpreted as conclusive evidence of adifference. FIG. 1 graphically illustrates the mean relative changes inFEV₁ % predicted from before to 30 minutes after dosing for each of thetreatments. TABLE 4 MEAN (SD) RELATIVE CHANGE IN FEV₁ % PREDICTED FEV₁ %TOBI 300 mg TOBI 30 mg TOBI 60 mg TOBI 90 mg Predicted PARI LC AerodoseAerodose Aerodose (%) PLUS¹ inhaler² inhaler² inhaler² Parameter (n =51) (n = 34) (n = 32) (n = 33) Predose 67.8 (18.4) 65.5 (17.1) 65.4(16.8) 71.3 (20.0) n = 51 n = 34 n = 32 n = 33 30 minutes 63.7 (17.6)63.0 (16.7) 62.5 (15.7) 68.7 (19.1) postdose n = 51 n = 34 n = 32 n = 32Relative −6.1 (5.2) −3.8 (5.4) −4.2 (6.2) −3.2 (7.4) change from n = 51n = 34 n = 32 n = 32 predose³ P-value for Treatment: Period: Carryover:crossover: 0.141 0.199 NC Pairwise 0.019 0.058 0.083 contrasts: C vs. Ep-value (paired t-test):

[0144] Safety Conclusions

[0145] Nine males and six females experienced treatment-inducedbronchospasm during the study. There was no difference in the rate ofoccurrence of TOBI induced bronchospasm between control and experimentaldelivery systems regardless of dose. The occurrence of bronchospasm wasrarely associated with patient symptoms. All but four of the patientsexperiencing drug-induced bronchospasm had been prescribedbronchodilators prior to the study suggesting that they had a history ofairway reactivity. The disproportionate number of males versus femalesexperiencing airway reactivity is unusual in light of the fact thatenrollment was approximately 60% female and 40% male. The pivotal trialsshowed that gender had no influence on drug induced airway reactivity.However, it would be difficult to base any conclusions on this findingdue to the small patient numbers in this study.

[0146] Treatment-emergent adverse events occurred in all treatmentgroups regardless of causality. The most common treatment-emergentexperiences were associated with Respiratory and Body as a Wholesystems. The most common individual events were cough increased,rhinitis, sputum increased, asthma, chest pain, and headache. Theseevents were also common to the patient's pretreatment symptomsreflecting the patients underlying disease. For the majority oftreatment-emergent adverse events, there were no meaningful differencesbetween TOBI doses or between the PARI LC PLUS nebulizer and theAeroDose™ inhaler.

[0147] The serious adverse events (SAEs) reported were primarilyassociated with an exacerbation of the patients underlying diseasestates. The one treatment-related SAE involved a possible sensitivityreaction that, if documented, would have occurred regardless of deviceor dose.

[0148] Review of the clinical chemistry, vital signs, and physicalfindings did not reveal any clinically significant safety issuesassociated with the dose or delivery system used to administer TOBI.

[0149] All the patients were on multiple concurrent medicationsappropriate to their disease state (cystic fibrosis), other underlyingillnesses, and age throughout the study. The concurrent medications didnot appear to have any influence on the safety profile of the study drugor either device during the study. Overall, no clinically significant orunexpected safety issues for TOBI were identified in the study. Thestudy showed that there were no meaningful differences in the safetyprofiles of administering TOBI via the PARI LC PLUS delivery system incomparison with the Aerodose delivery system regardless of dose.

[0150] Aerosol Delivery Results

[0151] Data Analysis

[0152] Forty-nine of the 52 dosed patients completed the study and wereevaluable for pharmacokinetics by reason of having completed at least 2doses of study treatments. These 49 patients also constituted the“completers” subset of patients referred to in the summary tables. Threeof the 52 dosed patients discontinued the study before completing 2doses of study treatments and were not evaluable for pharmacokinetics.All 52 patients were evaluable for the aerosol delivery objective(nebulization time) of the study.

[0153] Sputum Tobramycin Concentrations and Pharmacokinetic Parameters

[0154] Compliance with Specimen Collection Requirements

[0155] Six of 49 completing patients had a total of 11 missing sputumspecimens. No more than one sputum sample was missed per treatment-time(e.g., for TOBI 300 mg at one hour postdose). Two patients missed 2 ormore sputum samples during the study, and four patients missed a singlesputum sample.

[0156] A single completing patient provided no sputum pharmacokineticdata for the TOBI 60 mg treatment. One patient had missing sputumsamples from 10 minutes through 8 hours after TOBI 60 mg treatment.After the database was locked, the missing sputum concentration resultswere located. Sputum tobramycin concentrations at 10 minutes and 1, 2,4, and 8 hours were 0.82 μg/gm, BQL, 0.0, 0.0, and 0.0, respectively.The database was not subsequently unlocked to add these data, since theinclusion of these values would have had minimal impact on estimationand analyses of pharmacokinetic parameters. As a result, only C_(max)(0.82 μg/gm) and T_(max) (10 minutes=0.17 hour) values were excludedfrom TOBI 60 mg PK estimates and analyses; AUC values were incalculabledue to BQL tobramycin concentrations from one through 8 hours after TOBI60 mg treatment. Sputum Tobramycin Concentrations Pretreatment sputumtobramycin concentrations for all completing patients were below thelimit of quantifiability (LOQ) throughout the study.

[0157] After dosing, sputum concentrations increased rapidly, reachingmaximum concentrations within 10 minutes (see FIG. 2), and declinedthereafter with median half-life values ranging from approximately 1.6to 2.1 hours during the four treatments. The sputum concentrations werehighly variable among patients, as coefficients of variation (standarddeviation divided by the mean times 100%) approached or exceeded 100%for each treatment at all time points.

[0158] For the AeroDose™ inhaler, mean sputum tobramycin concentrationsincreased with increasing TOBI dose at each measurement time during the8-hour postdose period. Mean sputum concentrations for the TOBI 90 mgtreatment with the AeroDose™ inhaler were similar throughout the 8-hourperiod to those obtained for the TOBI 300 mg treatment with the PARI LCPLUS nebulizer.

[0159] By 2 hours after the end of TOBI 30 mg and by 8 hours after TOBI60 mg, 90 mg, and 300 mg treatments, sputum concentrations were belowLOQ in at least half of the patients. Period effects on sputumtobramycin concentrations were not observed.

[0160] After TOBI administration using the AeroDose™ inhaler, maximumplasma concentrations (C_(max)) and area under the plasma concentrationtime profile (AUC₀₋₈) increased linearly with dose (Table 5 below andFIGS. 3 and 4), suggesting linear pharmacokinetics. Dose normalizedC_(max) and AUC values were comparable among AeroDose™ dose levels,indicating dose proportionality (based on AUC values).

[0161] Comparing devices, mean C_(max) and AUC₀₋₈ for the TOBI 90 mgtreatment delivered by the AeroDose™ inhaler achieved similar levels asthose obtained by the TOBI 300 mg treatment delivered by the PARI LCPLUS nebulizer. The dose normalized C_(max). and AUC₀₋₈ results werehigher during AeroDose™ treatments than during the PARI LC PLUStreatment, indicating that the AeroDose™ inhaler exhibited higherefficiency. The bioavailability of the AeroDose™ device was about 3-foldhigher than that of the PARI LC PLUS nebulizer.

[0162] Exploratory analyses suggested that sputum pharmacokineticresults were unaffected by characteristics present before treatmentsbegan (age, gender, body weight, FEV₁ % predicted at screening) and wereunaffected by events noted after the start of treatments (devicefailure, occurrence of bronchospasm defined as a decrease ≧10% in FEV₁,and relative change in FEV₁ % predicted). TABLE 5 MEAN (SD) SPUTUMTOBRAMYCIN PHARMACOKINETIC PARAMETERS Sputum TOBI 300 mg TOBI 30 mg TOBI60 mg TOBI 90 mg Pharmaco- PARI LC Aerodose Aerodose Aerodose kineticPLUS^(a) inhaler^(b) inhaler^(b) inhaler^(b) Parameter (n = 49) (n = 34)(n = 32) (n = 32) C_(max) 985.65 329.05 577.83 958.00 (μg/gm) (839.34)(311.30) (538.42) (952.30) No. pts with 49 34 31 32 data: E vs C <0.0010.002 0.856 p-value^(c): E/C (0.23, 0.41) (0.43, 0.75) (0.72, 1.30) (90%CIs)^(d): Dose- 3.29 10.97 9.63 (8.97) 10.64 normalized (2.80) (10.38)(10.58) C_(max) (μg/gm)/mg No. pts with 49 34 31 32 data: E/C (2.82,5.13) (90% CIs)^(d): T_(max) (hr) 0.26 (0.38) 0.24 (0.24) 0.38 (0.76)0.33 (0.41) No. pts with 49 34 31 32 data: T_(½) (hr) 6.41 (24.09) 2.04(1.31) 12.89 13.02 (42.61) (36.91) Median T_(½) 1.71 1.78 2.06 1.60 (hr)No. pts with 41 15 21 24 data: AUC₀₋₈ 1471.16 360.79 804.78 1275.23 (hr· μg/gm) (1278.22) (422.23) (722.83) (1358.52) No. pts with 49 34 31 32data: E vs C <0.001 <0.001 0.465 p-value^(c): E/C (0.19, 0.28) (0.45,0.69) (0.72, 1.14) (90% CIs)^(d): Dose- 1.90 (4.26) 12.03 13.41 14.17normalized (14.07) (12.05) (15.10) AUC₀₋₈ (hr · μg/gm)/ mg No. pts with49 34 31 32 data: E/C (2.78, 4.12) (90% CIs)^(d): AUC_(0-∞) 1996.36638.68 1661.66 5544.88 (hr · μg/gm) (2013.70) (586.85) (2334.89)(14831.0) No. pts with 41 15 21 24 data:

[0163] Differences among the treatment groups in C_(max) and AUC₀₋₈(Table 5 above; FIGS. 3 and 4) were statistically significant (p<0.001)with no evidence of period or carryover (treatment by periodinteraction) effects. In pairwise comparisons, C_(max) and AUC₀₋₈ weresignificantly greater for TOBI 300 mg than for TOBI 30 mg and for TOBI60 mg but not for TOBI 90 mg (90% CIs for C_(max)=(0.72, 1.30); forAUC₀₈=(0.72, 1.14)).

[0164] The AeroDose™ inhaler was more efficient, regardless of TOBIdose, than the PARI LC PLUS nebulizer based on dose normalized sputumC_(max) and AUC₀₋₈ results. Dose normalized means for thesepharmacokinetic parameters were similar among AeroDose™ treatments butapproximately 3-fold higher than the dose normalized results after TOBI300 mg delivered by the PARI LC PLUS nebulizer (see Table 5).

[0165] The time to maximum sputum tobramycin concentrations (T_(max) inTable 5 above) was similar for all treatment groups and averaged between0.24 and 0.38 hours for AeroDose™ doses compared to 0.26 hours for theTOBI 300 mg treatment using the PARI LC PLUS. Elimination half-life(median T_(1/2) in Table 5) was also similar among AeroDose™ treatments,averaging 1.60 to 2.06 hours, compared to 1.71 hours for TOBI 300 mg.

[0166] Exploratory analyses revealed no substantial association betweensputum pharmacokinetic results and patient characteristics presentbefore treatments (age, gender, body weight, pulmonary function [FEV₁ %predicted] at screening) or emergent events after the start oftreatments (device failure, occurrence of bronchospasm [decrease≧10% inFEV₁ from predose to 30 minutes postdose], relative change in FEV₁.

[0167] Serum Tobramycin Concentrations and Pharmacokinetic Parameters

[0168] Forty-four (44) of 49 completing patients had no measurable serumtobramycin concentrations before dosing in any of the 3 treatmentperiods, and five patients exhibited measurable predose serum tobramycinabove the lower LOQ in the periods indicated in Table 6 below. TABLE 6MEASURABLE TOBRAMYCIN IN PREDOSE SERUM SPECIMENS Previous TreatmentPeriod Measurable^(b) Predose 8-hour Serum Tobramycin during TobramycinSerum Period Listed— Treatment^(a) TOBI Dose Concentration T_(1/2)Tobramycin Patient Sequence (mg) (μg/mL) (hr) Concentration (μg/mL)107-1030 C-1-2 prestudy na^(c) na^(c) Per 1—0.70 107-1027 C-1-3 300<0.20  1.68 Per 2—0.29 105-1034 C-2-1 prestudy na^(c) na^(c) Per 1—0.28300 1.00 7.75 Per 2—0.23 103-1019 1-C-2  30 0.35 10.85  Per 2—0.20102-1007 2-C-1 prestudy na^(c) na^(c) Per 1—0.77  60 0.75 7.71 Per2—1.38 300 0.96 10.62  Per 3—0.60

[0169] Table 6 also identifies predose serum specimens for periods 2, 3,or both that had measurable tobramycin in 4 of the 5 patients. Thesefindings are also reflected in non-zero mean amounts of predosetobramycin concentrations in periods 2 and 3. Three of the 5 patientsexhibited measurable serum tobramycin after having received TOBI 300 mgduring the immediately preceding study period.

[0170] These measurable predose results may represent carryover fromprevious treatment or non-specific assay interference, but the lowfrequency and magnitude of the results suggests that a substantialeffect on posttreatment analyses was unlikely.

[0171] After each of the four TOBI treatments, serum tobramycinconcentrations gradually increased, reaching a maximum at one hour afterdosing (FIG. 5), and declined thereafter with median half-lives rangingfrom 2.73 to 4.27 hours (Table 7 below).

[0172] For the Aerodose inhaler, mean serum tobramycin concentrationsincreased with increasing TOBI dose at each time during the 8-hourposttreatment period, but mean values for TOBI 90 mg were less at eachposttreatment time than those seen for TOBI 300 mg using the PARI LCPLUS nebulizer.

[0173] By 4 hours after the end of TOBI 30 mg and by 8 hours after TOBI60 mg and 90 mg treatments, serum concentrations were below LOQ in atleast half of the patients [median (50^(th) percentile) serumconcentrations=0.0 μg/mL]. More than half of the TOBI 300 mg patientsremained above the serum LOQ at 8 hours posttreatment. There was noapparent pattern of change in posttreatment serum tobramycinconcentrations from period to period for any of the 4 treatments, andthere was no clear indication of the presence of a carryover (treatmentby period interaction) effect in posttreatment results.

[0174] Serum Pharmacokinetic Parameters

[0175] After TOBI administration using the Aerodose inhaler, meanC_(max) and AUC results increased linearly with dose after theadministration of the 30, 60, and 90 mg doses (Table 7), suggestinglinear pharmacokinetics. Dose normalized AUC results were similar amongthe Aerodose dose levels, suggesting dose proportionality.

[0176] Comparing devices, C_(max) and AUC₀₋₈ for the TOBI 90 mg doseusing the Aerodose inhaler were not as high as results achieved by theTOBI 300 mg dose using the PARI LC PLUS nebulizer. However, thedose-normalized parameters were higher for the Aerodose inhaler at allthree TOBI dose levels, indicating better efficiency of the new device.Similar to the sputum data, the relative bioavailability wasapproximately 3-fold higher for the Aerodose inhaler as compared to thePARI nebulizer. The variability based on AUCs was similar for bothdevices.

[0177] Exploratory analyses suggested that serum pharmacokinetic resultswere unaffected by characteristics present before treatments began (age,gender, body weight, FEV₁ % predicted at screening) and were unaffectedby events noted after the start of treatments (device failure,occurrence of bronchospasm defined as a decrease ≧10% in FEV₁, andrelative change in FEV1 % predicted). TABLE 7 MEAN (SD) SERUM TOBRAMYCINCONCENTRATIONS BY TIME AND PHARMACOKINETIC PARAMETERS Serum TOBI 300 mgTOBI 30 mg TOBI 60 mg TOBI 90 mg Pharmaco- PARI LC Aerodose AerodoseAerodose kinetic PLUS^(a) inhaler^(b) inhaler^(b) inhaler^(b) Parameter(n = 49) (n = 34) (n = 32) (n = 32) C_(max) 1.12 (0.44) 0.38 (0.17) 0.69(0.34) 0.96 (0.40) (μg/mL) No. pts with 49 30 32 32 data: E vs C <0.001<0.001 0.027 p-value^(c): E/C (0.29, 0.36) (0.53, 0.66) (0.75, 0.96)(90% CIs)^(d): Dose- 0.0037 0.0127 0.0116 0.0106 normalized (0.0015)(0.0058) (0.0056) (0.0045) C_(max) (μg/mL)/mg No. pts with 49 30 32 32data: E/C (2.52, 3.25) (90% CIs)^(d): T_(max) (hr) 1.05 (0.38) 1.14(0.42) 0.98 (0.28) 1.14 (0.64) No. pts with 49 30 32 32 data: T_(½) (hr)3.42 (1.63) 6.75 (5.31) 4.16 (2.34) 3.10 (1.10) Median T_(½) 3.14 4.273.42 2.73 (hr) No. pts with 49 11 28 31 data: AUC₀₋₈ 4.96 (2.24) 1.43(1.43) 2.98 (1.92) 3.94 (1.52) (hr · μg/mL) No. pts with 49 30 32 32data: E vs C <0.001 <0.001 0.165 p-value^(c): E/C (0.18, 0.25) (0.46,0.62) (0.75, 1.03) (90% CIs)^(d): Dose- 0.0166 0.0478 0.0496 0.0438normalized (0.0075) (0.0477) (0.0319) (0.0169) AUC₀₋₈ (hr · μg/ mL)/mgNo. pts with 49 30 32 32 data: E/C (2.51, 3.21) (90% CIs)^(d): AUC_(0-∞)6.66 (4.32) 6.49 (7.71) 5.11 (4.62) 5.02 (1.63) (hr · μg/mL) No. ptswith 49 11 28 31 data:

[0178] Differences among treatment groups in serum C_(max) and AUC₀₋₈(Table 7 above; FIGS. 6 and 7) were statistically significant (p<0.001)with no period or carryover effects in the overall analyses. In pairwisecomparisons, C_(max) and AUC₀₋₈ were significantly greater for TOBI 300mg using the PARI LC PLUS than for TOBI 30 mg and TOBI 60 mg using theAerodose inhaler (p<0.001 in each comparison). C_(max) was statisticallysignificantly higher (p=0.027) for TOBI 300 mg compared to the TOBI 90mg dose, and AUC₀₋₈ was slightly but not significantly (p=0.165) greaterfor TOBI 300 mg than for TOBI 90 mg.

[0179] The Aerodose inhaler was more efficient, regardless of TOBI dose,than the PARI LC PLUS nebulizer based on dose normalized sputum C_(max)and AUC₀₋₈ results. Dose normalized means for these pharmacokineticparameters were similar among Aerodose treatments but approximately3-fold higher than the dose normalized results after TOBI 300 mgdelivered by the PARI LC PLUS nebulizer (Table 7).

[0180] T_(max) (Table 7) was similar for the four treatments, averagingbetween 0.98 and 1.14 hours for Aerodose treatments and 1.05 hours forthe TOBI 300 mg treatment using the PARI LC PLUS. Median T_(1/2) rangedfrom 2.73 to 4.27 hours among the Aerodose dose levels, compared to 3.14hours for TOBI 300 mg using the PARI LC PLUS nebulizer. Median T_(1/2)results using the Aerodose inhaler appeared to decrease with increasingTOBI dose, but this was considered an artifact related to greaterfrequency of missing T_(1/2) values (due to more BQL results) at lowerTOBI dose levels.

[0181] Exploratory analyses revealed no substantial association betweenserum pharmacokinetic results and patient characteristics present beforetreatments (age, gender, body weight, pulmonary function [FEV₁ %predicted] at screening) or emergent events after the start oftreatments (device failure, occurrence of bronchospasm [decrease≧10% inFEV₁ from predose to 30 minutes postdose], relative change in FEV₁.

[0182] Urinary Recovery of Tobramycin

[0183] Thirty-nine (39) of 49 completing patients had no measurableurine tobramycin concentrations before dosing in any of the 3 treatmentperiods, and 10 patients exhibited measurable predose urine tobramycinabove the lower LOQ in the periods indicated in Table 8 below. TABLE 8MEASURABLE TOBRAMYCIN IN PREDOSE URINE SPECIMENS Previous PeriodMeasurable^(b) Predose 8-24 hour Urine Tobramycin during TobramycinSerum Period Listed—Urine Treatment^(a) TOBI Dose Concentration T_(1/2)Tobramycin Patient Sequence (mg) (μg/mL) (hr) Concentration (μg/mL)103-1005 C-1-2 prestudy na^(d) na^(d) Per 1—3.80 300 3.92 4.80 Per2—2.06  30 2.48 not estimable Per 3—1.20 103-1039 C-1-3 prestudy na^(d)na^(d) Per 1—1.82 300 6.76 1.87 Per 2—2.58 104-1024 C-1-3 300 5.14 3.16Per 2—1.48 107-1027 C-1-3 prestudy na^(d) na^(d) Per 1—3.14 300 6.041.68 Per 2—1.58 104-1020 C-2-1 prestudy na^(d) na^(d) Per 1—1.74 30013.40  2.93 Per 2—2.28  60 5.80 12.96  Per 3—1.30 109-1014 C-2-3  60<1.0  4.06 Per 3—13.22 106-1025 1-C-2 300 5.14 3.80 Per 3—2.70 103-10122-C-3 300 2.26 3.63 Per 3—1.16 101-1002 3-C-1 300 7.82 3.37 Per3^(c)—1.12 103-1006 3-C-2 prestudy na^(d) na^(d) Per 1—2.72  90 10.10 3.14 Per 2—3.10 300 8.06 4.48 Per 3—2.08

[0184] Table 8 shows that measurable urine tobramycin was recoveredbefore dosing in periods 2, 3, or both for all 10 patients. Nine of the10 patients had measurable predose urine tobramycin after TOBI 300 mgtreatment during the preceding study period. One patient exhibitedmeasurable tobramycin in both predose serum and predose urine, and theseevents both followed TOBI 300 mg administration during the previousperiod.

[0185] Although carryover effect cannot be ruled out, the overallresults suggest that such an effect is unlikely. The eliminationhalf-life in sputum ranged from 1.60 to 2.06 hours, and in serum rangedfrom 2.73 to 4.27 hours, with no substantial differences between thefour treatments. Additionally, the amount of tobramycin excreted inurine was larger during the 0-8 hour period compared to the 8-24 hourperiod, consistent with the short T_(1/2) of tobramycin. Moreimportantly, in clinical Phase III studies in patients, multiple dailyadministrations did not result in any accumulation. Therefore it can beconcluded that such carryover effect is most likely due tononspecificity of the assay.

[0186] Consistent with the serum data, the amount of tobramycin excretedin urine was higher for TOBI 300 mg compared to TOBI 90 mg (Table 9below). However, the percent of dose excreted in urine was 3-fold higherfor the Aerodose inhaler at all dose levels (16 to 18%) as compared tothe PARI LC PLUS nebulizer. TABLE 9 MEAN (SD) URINARY RECOVERY OFTOBRAMYCIN BY TIME TOBI TOBI TOBI TOBI 300 mg 30 mg 60 mg 90 mg UrinePARI Aerodose Aerodose Aerodose Tobramycin LC PLUS^(a) inhaler^(b)inhaler^(b) inhaler^(b) Recovery (n = 49) (n = 34) (n = 32) (n = 32)Collection Interval Before and After Dosing: −12-0 hr predose 305.1122.8 67.9 615.5 (μg) (1412.0) (340.7) (192.8) (3202.5) No. pts withdata 48 33 32 31 0-8 hr postdose 15003.0 4835.6 8490.3 12304.8 (μg)(7116.2) (2649.6) (3159.6) (5352.7) No. pts with data 48 34 32 32Dose-normalized 50.0 161.2 141.5 136.7 (μg)/mg (23.7) (88.3) (52.7)(59.5) No. pts with data 48 34 32 32 E/C (90% CIs)^(d): (2.50, 3.62)8-24 hr postdose 3072.1 794.1 1367.4 2095.2 (μg) (2271.2) (853.1)(1118.8) (1818.7) No. pts with data 47 34 31 31 Dose-normalized 10.226.5 22.8 23.3 (μg)/mg (7.6) (28.4) (18.6) (20.2) No. pts with data 4734 31 31 E/C (90% CIs)^(d): (2.44, 3.48) Total 0-24 hour 18113.2 5629.79802.7 14588.1 (μg) (8303.4) (2993.6) (3771.0) (6044.9) No. pts withdata 46 34 31 31 Dose-normalized 60.4 187.7 163.4 162.1 (μg)/mg (27.7)(99.8) (62.8) (67.2) No. pts with data 46 34 31 31 E/C (90% CIs)^(d):(2.23, 3.27) Percent of Dose 6.0 18.8 16.3 16.2 Excreted (%)^(c)

[0187] For the Aerodose inhaler, mean 24-hour recovery of tobramycinfrom the urine increased with increasing TOBI dose during the study(Table 9 above; FIG. 8). Tobramycin recovery appeared to be doseproportional for the Aerodose inhaler, as mean 24-hour recoverynormalized for dose was similar among Aerodose treatments.

[0188] Comparing devices, mean recovery for the TOBI 90 mg treatment wasless than that seen for TOBI 300 mg using the PARI LC PLUS nebulizer.However, a greater percentage of the administered TOBI dose wasrecovered in the urine of patients who were dosed with the Aerodoseinhaler (18.8%, 16.3%, and 16.2%, respectively), irrespective of TOBIdose, than was recovered from patients who were dosed with the PARI LCPLUS nebulizer (6.0% of the administered TOBI 300 mg dose).

[0189] The largest amount of tobramycin was recovered during the first 8hours after dosing. There was no apparent pattern of period-to-periodchange in posttreatment urine tobramycin recovery for any of the 4treatments. Although a potential carryover could not be ruled out inapproximately 20% of the patients due to recovery of measurabletobramycin in predose urine, there was no clear indication of thepresence of a carryover (treatment by period interaction) effect inposttreatment results.

[0190] The percent of administered dose recovered in urine over 24 hourspostdose does not represent the delivered dose in the lung or absolutebioavailability. It is understood that a substantial amount of lungdeposited dose still remains in the body at 24 hours postdose.

[0191] Nebulization Time

[0192] Mean total nebulization time increased with increasing TOBI dose(Table 10 below; FIG. 9) and was substantially less when the Aerodoseinhaler was used at each TOBI dose level (mean ±SD for TOBI 30mg=2.8±1.0 min; TOBI 60 mg=5.4±2.1 min; TOBI 90 mg=8.0±2.5 min) thanwhen the PARI LC PLUS nebulizer was used (TOBI 300 mg=17.7±4.7 min).TABLE 10 MEAN (SD) NEBULIZATION TIME TOBI TOBI TOBI TOBI 300 mg 30 mg 60mg 90 mg PARI Aerodose Aerodose Aerodose LC PLUS¹ inhaler² inhaler²inhaler² Parameter (n = 51) (n = 34) (n = 32) (n = 33) Nebulization 17.7(4.7) 2.8 (1.0) 5.2 (2.1) 8.0 (2.5) Time³ (min) No. pts with data 51 3432 32

CONCLUSIONS

[0193] The Aerodose inhaler substantially reduced the amount of timerequired to nebulize the administered TOBI dose, compared to theapproved PARI LC PLUS nebulizer, and nebulization time increased withincreasing TOBI dose (TOBI 300 mg delivered by PARI LC PLUS mean=17.7minutes vs. 2.8 minutes, 5.4 minutes, and 8.0 minutes for TOBI 30 mg, 60mg, and 90 mg, respectively).

[0194] Sputum tobramycin concentrations throughout the 8-hour samplingperiod after dosing increased with increasing TOBI dose through 90 mgdelivered by the Aerodose inhaler, but results for TOBI 90 mg and TOBI300 mg delivered by the PARI LC PLUS nebulizer did not differsubstantially or consistently. Sputum tobramycin results were highlyvariable, with coefficients of variation approaching or exceeding 100%for each treatment at all time points. On average, sputum concentrationsreached their maximum at 10 minutes after each of the 4 treatments. By 2hours after TOBI 30 mg and by 8 hours after TOBI 60 mg, 90 mg, and 300mg, sputum concentrations were below the lower limit of quantifiability(LOQ) in at least half of the patients.

[0195] The mean of the maximum sputum concentration was significantlygreater after TOBI 300 mg (mean=985.65 μg/gm) than after TOBI 30 mg(329.05 μg/gm: p<0.001) and TOBI 60 mg (577.83 μg/gm: p=0.002) but notTOBI 90 mg (958.00 μg/gm: p=0.856; 90% CIs for the ratio of TOBI 90mg/TOBI 300 mg C_(max)=0.72, 1.30). The Aerodose inhaler was moreefficient than the PARI LC PLUS nebulizer based on sputum C_(max)results adjusted for TOBI dose administered (TOBI 300 mg with PARI LCPLUS: dose-normalized mean C_(max)=3.29 (μg/gm)/mg; TOBI 30, 60, and 90mg with Aerodose=10.97, 9.63, and 10.64 (μg/gm)/mg, respectively).

[0196] Mean sputum T_(max) was virtually identical for TOBI 300 mg(mean=0.26 hr) and TOBI 30 mg (0.24 hr) but was slightly less thanT_(max) for TOBI 60 mg (0.38 hr) and TOBI 90 mg (0.33 hr).

[0197] Mean sputum AUC₀₋₈ was significantly greater after TOBI 300 mg(mean=1471.16 hr·μg/gm) than after TOBI 30 mg (360.79 hr·μg/gm: p<0.001)and TOBI 60 mg (804.78 hr·μg/gm: p<0.001) but not TOBI 90 mg (1275.23hr·μg/gm: p=0.465; 90% CIs for the ratio of TOBI 90 mg/TOBI 300 mgAUC₀₋₈=0.72, 1.14). The greater efficiency of the Aerodose inhaler wasalso seen in dose-normalized AUC₀₋₈ results (TOBI 300 mg with PARI LCPLUS=4.90 [hr·μg/gm]/mg; TOBI 30, 60, and 90 mg with Aerodose=12.03,13.41, and 14.17 [hr·μg/gm]/mg, respectively).

[0198] No inferential analyses of sputum AUC_(0-∞) were performed due tohigh variability that increased with increasing TOBI dose.

[0199] Serum tobramycin concentrations also increased with increasingTOBI dose at each time during the 8-hour posttreatment observationperiod. Mean serum tobramycin concentrations reached their maximum atone hour after each treatment. By 4 hours after TOBI 30 mg and by 8hours after TOBI 60 mg and TOBI 90 mg, serum concentrations were belowLOQ in at least half of the patients. More than half of the TOBI 300 mgpatients remained above the serum LOQ at 8 hours posttreatment.

[0200] Mean serum C_(max) was significantly greater after TOBI 300 mg(mean=1.12 μg/mL) than after the other 3 treatments (TOBI 30 mg=0.38μg/mL, p<0.001; TOBI 60 mg=0.69 μg/mL, p<0.001; TOBI 90 mg=0.96 μg/mL,p=0.027). The Aerodose inhaler was also more efficient than the PARI LCPLUS nebulizer based on serum C_(max) results adjusted for TOBI doseadministered (TOBI 300 mg with PARI LC PLUS: dose-normalized meanC_(max)=0.0037 (μg/mL)/mg; TOBI 30, 60, and 90 mg with Aerodose=0.0127,0.0116, and 0.0106 (μg/mL)/mg, respectively.

[0201] Mean serum T_(max). was similar for the 4 treatments (mean=1.05hr, 1.02 hr, 0.98 hr, and 1.14 hr for TOBI 300 mg, 30 mg, 60 mg, and 90mg, respectively).

[0202] Mean serum AUC₀₋₈ was significantly greater after TOBI 300 mg(mean=4.96 hr·μg/mL) than after TOBI 30 mg (1.43 hr·μg/mL, p<0.001) andTOBI 60 mg (2.98 hr·μg/mL, p<0.001) but not TOBI 90 mg (3.94 hr·μg/mL,p=0.165; 90% CIs for the ratio of TOBI 90 mg/TOBI 300 mg AUC₀₋₈=0.75,1.03). The greater efficiency of the Aerodose inhaler was also seen indose-normalized AUC₀₋₈ results (TOBI 300 mg with PARI LC PLUS=0.0166[hr·μg/mL]/mg; TOBI 30, 60, and 90 mg with Aerodose=0.0478, 0.0496, and0.0438 [hr·μg/mL]/mg, respectively).

[0203] Serum AUC_((0-∞)) was not analyzed statistically due to highvariability but generally appeared to increase as the TOBI doseincreased.

[0204] Recovery of tobramycin from the urine within 24 hours afterdosing increased with increasing TOBI dose during the study (expressedin mg [mg=μg/1000], mean urine tobramycin recovery=18.1 mg, 5.6 mg, 9.8mg, and 14.6 mg after TOBI 300 mg, TOBI 30 mg, TOBI 60 mg, and TOBI 90mg doses, respectively). Most of the tobramycin was recovered within thefirst 8 hours after dosing. Normalized for dose, urine tobramycinrecovery within 24 hours was 6.0%, 18.8%, 16.3%, and 16.2% of theadministered TOBI 300 mg, TOBI 30 mg, TOBI 60 mg, and TOBI 90 mg doses,respectively.

[0205] Results of the present study showed that TOBI 300 mg delivered bythe PARI LC PLUS nebulizer (the control delivery system) and TOBI 30 mg,60 mg, and 90 mg delivered by the Aerodose inhaler (the experimentaldelivery system) were safe and well-tolerated by male and female cysticfibrosis patients. Fifteen patients (9 male and 6 female) experienced 24instances of bronchospasm (decline in FEV₁ (liters)>10%). There were nostatistically significant differences between control and anyexperimental treatment in the incidence of bronchospasm. There were nooverall treatment differences in quantitative change in FEV₁ frompredose to 30-minute postdose measurement times.

[0206] The study found no evidence that CF patients were at increasedrisk by reason of inhaling single TOBI doses of 30 mg, 60 mg or 90 mgcompared to the single TOBI 300 mg dose delivered by the PARI LC PLUSjet nebulizer. The most frequently reported treatment emergent adverseevents (cough increased, rhinitis, sputum increased, chest pain, asthma,and headache) and the SAEs reported by 4 of the patients were primarilyassociated with patients' underlying CF disease and related medicalconditions. The incidence of these events before and after studytreatments was substantially similar, suggesting that neither TOBI doselevels nor control and experimental inhalers altered ongoingsymptomatology associated with patients' underlying medical conditions.There were also no clinically significant safety issues reflected inclinical laboratory test results, vital signs, or physical findings.

[0207] In this example, the Aerodose inhaler substantially reduced thetime required for nebulization of all three dose levels (30 mg, 60 mg,and 90 mg) of TOBI compared to the nebulization time for the approvedTOBI 300 mg delivery system using the PARI LC PLUS jet nebulizer.Average nebulization times were 2.8, 5.4, and 8.0 minutes using theAerodose inhaler to deliver TOBI 30 mg, 60 mg, and 90 mg, respectivelyvs. 17.7 minutes using the PARI LC PLUS nebulizer to deliver TOBI 300mg. The Aerodose inhaler therefore cut nebulization time of the TOBI 90mg dose by more than 50% compared to the PARI LC PLUS nebulizer in thepresent study, and nebulization times for lower TOBI doses were reducedby even greater amounts. Present nebulization time results in CFpatients >12 years of age with baseline FEV₁ % predicted >40% wereconsistent with those obtained after single doses of TOBI 60 mg usingthe Aerodose inhaler (mean=5.7 minutes) but slightly less than TOBI 300mg results using the PARI LC PLUS nebulizer (mean=20.4 minutes) in theTOBI gamma scintigraphy study of tobramycin deposition in the lungs ofhealthy adult male and female volunteers of Example 2, infra.

[0208] This example demonstrates that TOBI 90 mg (but not TOBI 60 mg orTOBI 30 mg) delivered by the Aerodose inhaler achieved similar actualpulmonary deposition, systemic absorption, and urinary recovery oftobramycin as that achieved by administration of the TOBI 300 mg dosedelivered by the PARI LC PLUS nebulizer. Normalized for TOBI dose, theAerodose inhaler was substantially more efficient than the PARI LC PLUSnebulizer in the delivery of aerosolized tobramycin to the lungs and tothe systemic circulation.

[0209] Pulmonary deposition of tobramycin was measured by determinationof sputum tobramycin concentrations and by calculation of sputumpharmacokinetic parameters. Maximum sputum tobramycin concentrationswere reached by 10 minutes after administration of each treatment, andconcentrations were below the LOQ in half or more of the patients at 2hours after TOBI 30 mg and at 8 hours after TOBI 60 mg, 90 mg, and 300mg. The extent of pulmonary deposition of tobramycin, as measured bymaximum sputum concentrations and sputum AUC₀₋₈ results, increased withincreasing TOBI dose through 90 mg, but TOBI 90 mg and TOBI 300 mg didnot differ statistically (mean sputum C_(max)=958.00 and 985.65 μg/gm;mean sputum AUC₀₋₈=1275.23 and 1471.16 hr·μg/gm, respectively). Meansputum C_(max) results after TOBI 30 mg and 60 mg doses weresignificantly less than that of the TOBI 300 mg dose. Present sputumC_(max) results achieved after the single TOBI 300 mg dose were slightlyless than sputum tobrarnycin concentrations achieved 10 minutes after asingle TOBI 300 mg dose (mean sputum tobramycin concentration=1237μg/gm, median=1090 μg/gm) in two large previously conducted Phase IIIpivotal trials.

[0210] The results of this example demonstrate that at least one of thethree TOBI doses (TOBI 90 mg) delivered by the experimental Aerodoseinhaler achieved similar actual sputum tobramycin concentrations andthat these results in turn were similar to sputum results obtained inthe prior pivotal studies supporting the commercial TOBI product. It isalso important that present sputum results demonstrated that theexperimental Aerodose inhaler was substantially more efficient,regardless of TOBI dose, in delivery of aerosolized tobramycin to thelung than the PARI LC PLUS jet nebulizer. Dose-normalized sputum C_(max)was 10.97, 9.63, and 10.64 (μg/gm)/mg for TOBI 30 mg, 60 mg, and 90 mgdelivered by Aerodose inhaler, respectively, compared to 3.29 (μg/gm)/mgfor TOBI 300 mg delivered by PARI LC PLUS. Similarly, dose-normalizedsputum AUC₀₋₈ was 12.03, 13,41, and 14.17 [hr·μg/gm]/mg for TOBI 30 mg,60 mg, and 90 mg delivered by Aerodose inhaler, respectively, comparedto 4.90 [hr·μg/gm]/mg for TOBI 300 mg delivered by PARI LC PLUS.

[0211] Systemic absorption of tobramycin was measured by determinationof serum tobramycin concentrations and by calculation of serumpharmacokinetic parameters. Maximum serum tobramycin concentrations werereached at one hour after each of the four TOBI treatments, andconcentrations were below LOQ in half or more of the patients by 4 hoursafter TOBI 30 mg and by 8 hours after TOBI 60 mg and 90 mg. More thanhalf of the patients at TOBI 300 mg had measurable serum tobramycin at 8hours postdose. The extent of absorption of tobramycin, as measured byserum C_(max) results, increased with increasing TOBI dose, as C_(max)was significantly greater after TOBI 300 mg (mean=1.12 μg/mL) than aftereach of the lower TOBI doses (mean=0.38, 0.69, and 0.96 μg/mL for TOBI30 mg, 60 mg, and 90 mg doses, respectively). Serum C_(max) for TOBI 300mg in the present study was slightly higher (mean ±SD=1.10±0.44 μg/mLwith a mean T_(max) of 1.05 hr) than the mean serum tobramycinconcentration reported at one hour after TOBI 300 mg in the TOBI NDA(0.95±0.50 μg/mL). Serum C_(max) achieved by the Aerodose inhaler at theTOBI 90 mg dose level in the current study was virtually identical tothe NDA serum concentrations one hour after TOBI 300 mg (mean=0.96±0.37μg/mL), although it was significantly (p=0.027) less than the currentTOBI 300 mg.

[0212] Thus, present serum tobramycin results demonstrated that TOBI 90mg delivered by the Aerodose inhaler were similar (AUC₀₋₈) or nearlysimilar (C_(max)) to those obtained after TOBI 300 mg delivered by thePARI LC PLUS nebulizer in the present study and in the prior pivotalstudies supporting the TOBI commercial product. Present serum resultsalso demonstrated that the experimental Aerodose inhaler wassubstantially more efficient, regardless of TOBI dose, in delivery ofaerosolized tobramycin to the systemic circulation than the PARI LC PLUSjet nebulizer. Dose-normalized serum C_(max) was 0.0127, 0.0116, and0.0106 (μg/mL)/mg for TOBI 30 mg, 60 mg, and 90 mg delivered by Aerodoseinhaler, respectively, compared to 0.0037 (μg/mL)/mg for TOBI 300 mgdelivered by PARI LC PLUS. Similarly, dose-normalized serum AUC₀₋₈ was0.0478, 0.0496, and 0.0438 [hr·μg/mL]/mg for TOBI 30 mg, 60 mg, and 90mg delivered by Aerodose inhaler, respectively, compared to 0.0166[hr·μg/mL]/mg for TOBI 300 mg delivered by PARI LC PLUS. The greaterefficiency of the Aerodose inhaler observed in present serum tobramycinresults is consistent with greater efficiency and less wastage of thetobramycin dose observed in earlier studies.

[0213] Urinary recovery of tobramycin was measured by determining thecumulative amount of tobramycin recovered in urine collected for 24hours after dosing. The amount of urinary tobramycin recovered within 24hours postdose increased with increasing TOBI dose (expressed in mg[mg=μg/1000], mean urine tobramycin recovery=5.6 mg, 9.8 mg, 14.6 mg,and 18.1 mg tobramycin after TOBI 30 mg, 60 mg, 90 mg, and 300 mg). Theresults were not tested statistically, and it was not possible todetermine whether TOBI 90 mg and TOBI 300 mg results for 24-hourrecovery of urine tobramycin were similar or different.

[0214] Normalized for dose by dividing the mean amount of tobramycinrecovered by the nominal amount of TOBI administered, urinary recoveryof tobramycin was approximately 18.8%, 16.3%, 16.2%, and 6.0% of theadministered TOBI 30 mg, 60 mg, 90 mg, and 300 mg doses, respectively.

[0215] During the study, measurable tobramycin (i.e., above the lowerlimit of quantifiability [LOQ] of the assay) was detected in 12-hourpredose urine collections in a total of 10 patients, including 5patients before the first dose of study treatments in period one and all10 patients before the second or third doses in periods 2 and 3 or both.Similarly, measurable tobramycin was detected in predose serum specimensin a total of 5 patients, including 3 patients before the first dose ofstudy treatments in period one and 4 patients before dosing in periods2, or 3, or both. A single patient had measurable tobramycin in bothurine and serum.

[0216] Substantial variability is known to occur among patients in therate and extent of uptake, renal accumulation, and elimination ofaminoglycoside antibiotics, even in patients with normal renal function.Each of these factors may lengthen the amount of time that measurableconcentrations of aminoglycoside antibiotics may be detected in serumand urine. The present study employed a prestudy washout interval of 7days from previous prescription aminoglycoside antibiotic use and a7-day interval between the 3 single doses of TOBI, an aminoglycosideantibiotic, during the crossover treatment periods. It is plausible thatprestudy and on-study washout intervals in the study may have been tooshort for complete elimination of residual tobramycin previouslyadministered, if any. Measurable amounts of tobramycin for thesepatients would have had little effect on study results, since theamounts and concentrations detected were very small in nearly all cases,and no unusually high serum or urine tobramycin results were notedduring the study.

[0217] The Aerodose inhaler was a safe and efficient aerosolization anddelivery device for TOBI during the study.

Example 2 Scintigraphy Study

[0218] In order to assess the in vivo lung deposition of 300 mgtobramycin (TOBI®) inhaled using the PARI LC PLUS™ jetnebulizer/DeVilbiss PulmoAide® compressor delivery system (currentcommercial delivery system) compared with the deposition of 60 mgtobramycin (TOBI®) using the AeroDose™ inhaler in accordance with thepresent invention, a gamma scintigraphy study was performed. The imagingtechnique of gamma scintigraphy is a well-established method¹⁰⁻¹² thatprovides precise quantification of drug delivered to the lungs¹³. Italso provides an assessment of the distribution of deposited drug indifferent lung regions (peripheral, intermediate and central lungregions corresponding to small airways, medium sized airways and largeairways, respectively¹⁴). Gamma scintigraphy is the only non-invasivemethod currently available for obtaining this type of information.

[0219] The study of this example was designed as an open label,randomized, single center, single dose, two period crossover Phase Istudy of aerosol delivery characteristics and safety of two inhalationdevices in healthy adult volunteers. A maximum of 14 healthy male ornon-pregnant, non breast-feeding female volunteers aged 18 to 65 yearsof age were to receive in random order two single doses of aerosolizedantibiotic mixed with a sterile radiotracer (technetium bound todiethylenetriaminepentaacetic acid: ^(99m)Tc DTPA) separated by awashout interval of a minimum of 44 hours between doses. Radiolabeledaerosols consisted of a single 300 mg dose in a 5 mL solution of TOBIdelivered by the control delivery system (PARI LC PLUS jet nebulizerwith a PulmoAide compressor) and a single 60 mg dose in a 1 mL solutionof TOBI delivered by the experimental delivery system (Aerodoseinhaler).

[0220] Aerosol delivery characteristics of control and experimentaldelivery systems were compared on the basis of lung deposition ofradiolabeled tobramycin determined by gamma scintigraphy, time tocomplete nebulization of aerosolized doses, serum concentrations oftobrarnycin determined by Abbott TDxFLx assays, and serum tobramycinpharmacokinetic parameters.

[0221] The safety of control and experimental TOBI delivery systems wascompared on the basis of changes in pulmonary function, the incidence oftreatment emergent adverse events, and the occurrence of clinicallysignificant laboratory and clinical evaluations and of unusually highserum tobramycin concentrations.

[0222] The duration of study participation for each subject was to beapproximately five weeks including a screening period of up to 3 weeksin duration, two treatment periods of approximately 9 hours eachseparated by a minimum 44-hour washout interval, and a follow-up periodthrough 2 weeks after the end of dosing.

[0223] Treatments

[0224] TOBI® was administered by inhalation as a single 300 mg dose andas a single 60 mg dose to each subject during the study. The 300 mg dosewas supplied as a commercial ampoule of TOBI. The 60 mg dose oftobramycin solution was prepared by study site personnel by withdrawing1.0 mL of solution from the 300 mg/5 mL commercial ampoule of TOBI intotwo unit dose syringes containing 0.5 mL each.

[0225] Sterile ^(99m)Tc DTPA was added as a radiotracer to both 300 mgand 60 mg solutions at the study site prior to instillation into thenebulizer. Sufficient ^(99m)Tc DTPA was added to both the 300 mg and the60 mg dose so that no more than 10 MBq ^(99m)Tc DTPA was delivered tothe subject with each single dose administered.

[0226] Using control and experimental aerosol delivery systems, eachsubject was to self-administer two single aerosolized doses ofradiolabeled (^(99m)Tc DTPA) TOBI, one dose in each of two crossovertreatment periods, according to the randomization scheme for the study.Subjects were instructed to use nose clips and breathe in a normalbreathing pattern while inhaling the medication according to theinstructions for use for each inhaler.

[0227] Control and experimental treatment delivery systems werespecified as follows.

[0228] Control Treatment Delivery System: PARI LC PLUS jet nebulizerwith DeVilbiss PulmoAide compressor delivering 300 mg (5 mL) of TOBI.

[0229] Experimental Treatment Delivery System: Aerodose inhalerdelivering 60 mg (1 mL) of TOBI.

[0230] When the PARI LC PLUS nebulizer was used, 5 mL radiolabeled TOBIwas added to the drug reservoir and nebulized without interruption untilthe nebulizer reservoir was dry. The PARI system was configured suchthat exhalation by the subject did not result in escape of radioactiveaerosol into the surrounding atmosphere. Exhaled droplets were collectedusing a filter attached to the side of the inhaler by a T-piece. Inaddition, a scavenger filter was placed above the inhaler, which was inturn connected to a vacuum pump. The scavenger system was used tocollect any radiolabeled droplets escaping from the inhaler.

[0231] When the Aerodose inhaler was used, one 0.5 mL aliquot ofradiolabeled TOBI was added to the drug reservoir and nebulized todryness. A second 0.5 mL dose was then added to the reservoir andnebulized to dryness. The inhaler was surrounded with an exhaled aircollection box. Air was drawn through a filter at the back of the boxusing a vacuum pump.

[0232] Start and stop times of nebulization for both the Aerodose andPARI LC PLUS nebulizers were to be recorded in CRFs. Nebulization timefor the Aerodose inhaler was not to include the time needed to refillthe drug reservoir according to the protocol.

[0233] Enrolled volunteers were randomly assigned to two treatmentsequence groups as illustrated below according to a randomizationscheme.

[0234] PARI 300 mg/Aerodose 60 mg:

[0235] period 1: PARI LC PLUS with TOBI 300 mg

[0236] period 2: Aerodose with TOBI 60 mg

[0237] Aerodose 60 mg/PARI 300 mg:

[0238] period 1: Aerodose with TOBI 60 mg period 2: PARI LC PLUS withTOBI 300 mg

[0239] All subjects randomly assigned to a single treatment sequencegroup received control and experimental treatments in the same orderduring the study, while subjects assigned to the other treatmentsequence group received treatments in the reverse order. Table 11 belowshows the two sequences of treatment administration employed during thestudy via the randomization process. TABLE 11 TREATMENT SEQUENCE GROUPSAND SEQUENCE OF TREATMENTS IN THE STUDY Treatment Sequence Group¹Treatment Period 1 Treatment Period 2 C-E² C E E-C E C

[0240] Before dosing, TOBI formulations were radiolabeled with^(99m)Tc-DTPA in preparation for gamma scintigraphy to determineposttreatment tobramycin deposition in the lungs. Subjects practiced theinhalation procedure with both control and experimental devices filledwith normal saline. When the investigator was satisfied that the subjectcould reproducibly perform the correct inhalation technique, the inhalerwas filled with the radiolabeled formulation, and the subject inhaledthe radiolabeled dose until the nebulizer was dry and nebulization wasstopped.

[0241] Immediately following inhalation of radiolabeled, aerosolizedtobramycin, scintigraphic images were recorded to determineradioactivity associated with lung and oropharyngeal tobramycindeposition and with external items such as nebulizer parts, mouthpieces,filters, and tissues used by subjects. If not previously done within thelast 5 years, a posterior lung ventilation scan was also performedduring the study after subjects inhaled the radioactive inert gas^(81m)Kr to determine the lung outlines and facilitate the determinationof regional deposition of radiolabeled tobramycin.

[0242] Deposition of Tobramycin

[0243] Assessment and comparison of tobramycin deposition patternsbetween PARI LC PLUS and Aerodose delivery systems was a primaryobjective of the study. Deposition patterns of inhaled, radiolabeledtobramycin were determined using scintigraphic imaging methodology.Lung, oropharyngeal, and (if necessary) abdominal radioactivity wasmeasured from images obtained immediately after inhalation of eachsingle dose of radiolabeled tobramycin using a gamma camera (GeneralElectric Maxicamera) with a 40 cm field of view and fitted with a lowenergy parallel hole collimator. Images were obtained as describedbelow:

[0244] posterior view of the chest;

[0245] anterior view of the chest;

[0246] right lateral view of the oropharynx;

[0247] anterior and posterior abdominal views if necessary, i.e., ifactivity had spread through the intestine, beyond the field of view ineither of the chest images;

[0248] items external to the body of the subject as follows:

[0249] for the PARI LC PLUS system:

[0250] nebulizer cup

[0251] mouthpiece

[0252] exhalation filter and T-piece

[0253] scavenger filter

[0254] any tissues used by the subject

[0255] for the Aerodose system:

[0256] Aerodose inhaler

[0257] exhaled air containment box and filter

[0258] any tissues used by the volunteer

[0259] Additionally, a posterior lung ventilation scan was performedusing the radioactive inert gas, krypton (^(81m)Kr), to determine thelung outlines. The lung outlines were used to divide lung images of eachsubject into central, intermediate, and peripheral lung zones in orderto determine the amount of aerosolized tobramycin deposited in each ofthese zones¹⁷. Lung ventilation scans taken for subjects whoparticipated in earlier studies were acceptable for use for this studyprovided the scan was obtained within the last five years and thesubject had no record of serious lung disease in the intervening period.

[0260] Deposition zones of interest on scintigraphic images wereadditionally drawn around the oropharynx, esophagus, and stomach(including any activity in the small intestine). The counts obtainedwithin all regions of interest were corrected for backgroundradioactivity, radioactive decay, and for tissue attenuation¹⁸. Inregions where both anterior and posterior images were recorded, thegeometric mean of counts in both images was calculated prior tocorrection for tissue attenuation. Determination of the percentage ofthe dose deposited in the oropharynx included activity adhering to themouth and oropharynx together with any swallowed activity detected inthe esophagus, stomach, and intestine.

[0261] All images were recorded using Micas X plus software installed ona UNIX based computer system. Images were stored on digital audio tape(DAT) for subsequent analysis and archiving. Scintigraphic data wereanalyzed by Pharmaceutical Profiles Ltd. (PPL) in accordance with thePPL Standard Operating Procedure N 1013 “Lung Quantitative DataAnalysis”. The data were summarized to obtain the following parameters:

[0262] whole lung deposition (% of metered dose);

[0263] central lung zone deposition (% of metered dose);

[0264] intermediate lung zone deposition (% of metered dose);

[0265] peripheral lung zone deposition (% of metered dose);

[0266] ratio of peripheral to central zone deposition (lung penetrationindex);

[0267] oropharyngeal deposition (including esophagus and stomach) (% ofmetered dose);

[0268] inhaler deposition (PARI LC PLUS or AeroDose) (% of metereddose);

[0269] radioaerosol in exhaled air (filters) (% of metered dose);

[0270] radioaerosol on PARI LC PLUS mouthpiece, T-piece, scavengerfilter and subject tissues (% of metered dose);

[0271] radioaerosol on Aerodose exhaled air collection box and subjecttissues (% of metered dose).

[0272] The counts in each area were expressed as a percentage of themetered dose that was determined from the sum of the total body countsin addition to those deposited on the inhaler and the exhalation filter.Since the volume of TOBI placed into each of the two inhalers wasdifferent, direct comparisons of the percentage deposition values wasproblematic. To aid interpretation of the data, the percentagedeposition values were multiplied by the nominal metered dose (300 mgfor the PARI LC PLUS and 60 mg for the Aerodose inhaler) to obtainamounts of drug deposited in milligrams for each of the depositionparameters listed above.

[0273] Nebulization Time

[0274] Assessment and comparison of nebulization time between PARI LCPLUS and Aerodose delivery systems was another objective of the study.Elapsed time from the start of nebulization (defined as the subject'sfirst tidal breath after the inhaler was in place) until no more TOBIsolution was aerosolized by the inhaler was measured by staff at thesite using a stopwatch. Nebulization time was not to include time neededfor instillation of drug into the nebulizer between the repeat fillingof the Aerodose inhaler. The length of any interruption in nebulizationand the reason for interruption were recorded.

[0275] Serum tobramycin concentrations were determined for the presentstudy, and pharmacokinetic parameters were calculated, to providepreliminary estimates of the bioavailability of 60 mg TOBI delivered bythe Aerodose system in comparison with that of the marketed 300 mg TOBIformulation. Additionally, unusually high serum tobramycin results (≧4μg/mL) were considered an important measure of safety during the study.

[0276] Venous blood samples (8 mL) for the determination of serumtobramycin concentrations were collected by intravenous cannula or byvenipuncture before each single dose of TOBI and at 30 minutes and 1, 2,4, and 8 hours after completion of dosing. The first one mL of bloodwithdrawn from the cannula was discarded, and the subsequent 7 mL waswithdrawn into serum sampling tubes. Cannulae were frequently flushedwith saline during the course of the treatment day.

[0277] Blood samples were centrifuged at approximately 1600 g for 10minutes at 4° C. The resulting serum fraction was split into twoaliquots by pipetting into two prelabeled polypropylene screw cap tubes.Tubes were stored at −20° C. for each study period and were thentransferred to a −70° C. freezer.

[0278] The maximum tobramycin concentration (C_(max)) and the time toreach C_(max) (T_(max)) were the observed values. The elimination rateconstant (k_(el): used to calculate AUC_(0-∞); see next paragraph) wascalculated as the negative slope of the log plasma concentration vs.time plot using the last two measurable concentrations. Use of more thantwo concentrations at or after T_(max) is preferred for calculation ofthe elimination rate constant; however, several subjects had only twomeasurable tobramycin concentrations at the terminal phase after TOBI 60mg using the Aerodose inhaler. The alternate method of calculatingk_(el) using the last two measurable concentrations was employed for allsubjects for both period 1 and period 2.

[0279] Area under the curve through 8 hours postdose (AUC₀₋₈) andextrapolated to infinity (AUC_(0-∞)) were calculated for serumtobramycin concentrations using the linear trapezoid rule. Actualnebulization time was added to the time between predose and 30 minutesafter the end of inhalation when calculating AUC₀₋₈. AUC_(0-∞) wasextrapolated from the last measurable concentration to infinite time byadding the quantity equal to the last measurable concentration dividedby the elimination rate constant (k_(el)).

[0280] Statistical Methods Planned in the Protocol

[0281] Scintigraphic data were analyzed in accordance with the currentversion of the PPL Standard Operating Procedure N 1013 “LungQuantitative Data Analysis”. Manipulation and calculation ofradioactivity counts were accomplished using a custom written region ofinterest program, where regions of interest were central, intermediate,peripheral, and stomach/intestines if necessary. Numerical data weredownloaded automatically from the Park Medical computer into acustomized spreadsheet.

[0282] Due to the small number of subjects in the study, statisticalanalysis was performed only on whole lung deposition data and onselected pharmacokinetic parameters. All other study data weresummarized descriptively. Descriptive summaries for quantitative dataincluded sample size, mean, standard deviation, median, minimum,maximum, and/or range values as appropriate. Descriptive summaries forqualitative or categorical data included number and percent of subjectswith the characteristic. All clinical data manipulations, analyses,summaries, and transformations employed SAS version 6.12²⁰⁻²².

[0283] Aerosol Delivery Analyses

[0284] Whole lung deposition was the primary endpoint for the analysis.The Wilcoxon one-sample, matched-pairs, signed ranks test was used todetermine whether differences between the whole lung deposition patterns(percent and amount of metered dose deposited) for the two inhalers weresignificant. The significance level was set at α=0.05.

[0285] Serum pharmacokinetic parameters (C_(max), AUC₀₋₈, and AUC_(0-∞))were analyzed for differences between delivery systems using a repeatedmeasures analysis of variance. The statistical model included studyperiod and delivery systems as fixed effects and subject as the randomeffect. The carryover effect from treatment period 1 to 2 was alsoinvestigated. The significance level was set at α=0.05, and tests ofsignificance were two-sided.

[0286] Additional deposition measures of interest, nebulization time,serum tobramycin concentrations and pharmacokinetic parameters weresummarized and evaluated descriptively for apparent differences betweenaerosol delivery systems.

[0287] Study Drug Administration

[0288] All subjects were successfully dosed according to therandomization schedule for the study, and all subjects received andcompleted both inhalation administrations. All subjects received singledoses of TOBI 300 mg and TOBI 60 mg during the study.

[0289] Deposition of Radiolabeled Tobramycin

[0290] Tobramycin deposition results indicated that the Aerodose systemwas more efficient than the PARI LC PLUS system. The Aerodose systemwith TOBI 60 mg delivered a greater percentage of the dose to the lungs(mean ±SD=34.8±10.1%) than the PARI system with TOBI 300 mg (8.2±3.6%),and the difference was statistically significant (p=0.005) (see Table 12below). Results from the analysis (n=9) that excluded data from onepatient were similar (means=35.4% vs. 9.1% for Aerodose and PARIsystems, respectively; p=0.008).

[0291] The actual amount of drug delivered to the lungs (Table 13 below)was slightly but not significantly less (p=0.202) using the Aerodoseinhaler (20.9±6.0 mg) than using the PARI inhaler (24.5±10.7 mg).Excluding subject 1007, the analysis showed significantly less (p=0.04)deposition of the Aerodose 60 mg dose (21.2 mg) than the PARI 300 mgdose (27.2 mg). TABLE 12 MEAN (SD) PERCENTAGE DEPOSITION OF THE METEREDTOBI DOSE Intent to Treat Excluding Subject 1007 (n = 10) (n = 9) TOBITOBI 300 mg TOBI 300 mg TOBI Zone of PARI LC 60 mg PARI LC 60 mgDeposition PLUS AeroDose PLUS AeroDose p-value* Whole lung 8.2 (3.6)*34.8 (10.1)* 9.1 (2.2) 35.4 (10.5) 0.005 central 2.4 (1.2) 10.1 (4.0)2.7 (0.9) 10.2 (4.2) intermediate 2.7 (1.2) 11.6 (3.6) 3.0 (0.8) 11.8(3.7) peripheral 3.1 (1.3) 13.2 (3.4) 3.5 (0.7) 13.4 (3.5) ratio:peripheral/central 1.2 (0.5) 1.4 (0.4) 1.4 (0.3) 1.4 (0.4) Oropharynx(including 14.4 (6.7) 31.5 (11.6) 16.0 (4.7) 31.5 (12.3) esophagus andstomach) Inhaler 42.6 (6.7) 15.2 (8.4) 43.5 (6.4) 15.1 (8.9) Exhalationfilter 31.6 (10.9) 16.9 (5.6) 28.3 (2.7) 16.3 (5.6) PARI-specific:mouthpiece 1.0 (0.5) 1.0 (0.5) T-piece 2.0 (0.6) 2.0 (0.5) tissue 0.0(0.1) 0.0 (0.1) scavenger filter 0.1 (0.1) 0.1 (0.1) AeroDose-specific:box 1.7 (1.5) 1.6 (1.6) tissue 0.0 (0.1) 0.0 (0.1)

[0292] The Aerodose inhaler deposited proportionally more (Table 12above) tobramycin in the lungs than in the oropharynx (mean 34.8% vs.31.5% of the 60 mg dose), while the PARI LC PLUS nebulizer depositedless tobramycin in the lungs than in the oropharynx (mean 8.2% vs. 14.4%of the 300 mg dose). The ratio of lung to oropharyngeal deposition(whole lung deposition divided by oropharynx deposition in Table 12above) was approximately 1.1 for the Aerodose inhaler compared toapproximately 0.6 for the PARI LC PLUS nebulizer.

[0293] Regional deposition within the lung was predominantly peripheraland very similar for both inhalers (ratio of radioactivity in peripheralto central zones: Aerodose=1.4±0.4; PARI LC PLUS=1.2±0.5).

[0294] Substantially less tobramycin was deposited on the Aerodoseinhaler (15.2±8.4%; 9.1±5.1 mg;-Tables 4 and 5, respectively) andexhalation filter (16.9±5.6%; 10.1±3.3 mg) than on the PARI LC PLUSnebulizer (42.6±6.7%; 127.8±20.0 mg) and filter (31.6±10.9%; 94.8±32.7mg). No more than 2% of the metered dose was deposited oninhaler-specific surfaces or tissue paper used by subjects. TABLE 13MEAN (SD) AMOUNT (MG) OF DEPOSITION OF THE METERED TOBI DOSE Intent toTreat Excluding Subject 1007 (n = 10) (n = 9) TOBI 300 mg TOBI 300 mgZone of PARI LC TOBI 60 mg PARI LC TOBI 60 mg Deposition PLUS AeroDosePLUS AeroDose p-value* Whole lung 24.5 (10.7)* 20.9 (6.0)* 27.2 (6.7)21.2 (6.3) 0.202 central 7.3 (3.6) 6.0 (2.4) 8.0 (2.8) 6.1 (2.5)intermediate 8.0 (3.7) 6.9 (2.1) 8.9 (2.5) 7.1 (2.2) peripheral 9.3(3.8) 7.9 (2.1) 10.4 (2.0) 8.1 (2.1) Oropharynx (including 43.3 (20.2)18.9 (6.9) 48.1 (14.0) 18.9 (7.4) esophagus and stomach) Inhaler 127.8(20.0) 9.1 (5.1) 130.5 (19.2) 9.0 (5.4) Exhalation filter 94.8 (32.7)10.1 (3.3) 84.8 (8.1) 9.8 (3.4) PARI-specific: mouthpiece 3.0 (1.4) 3.1(1.5) T-piece 6.1 (1.7) 5.9 (1.6) tissue 0.1 (0.2) 0.1 (0.2) scavengerfilter 0.4 (0.4) 0.4 (0.4) AeroDose-specific: box 1.0 (0.9) 1.0 (0.9)tissue 0.0 (0.1) 0.0 (0.1)

[0295] Nebulization Time

[0296] The nebulization time (i.e., time required from first tidalbreath until the nebulizer ran dry) was significantly shorter (p=0.005)for the Aerodose delivery system (mean ±SD=5.70±1.16 minutes) than forthe PARI LC PLUS system (20.40±3.47 minutes) (Table 14 below). TABLE 14MEAN (SD) NEBULIZATION TIME Intent to Treat (n = 10) Nebulization Time*TOBI 300 mg TOBI 60 mg Parameter PARI LC PLUS AeroDose p-valueNebulization Time (minutes): Mean 20.40 5.70 0.005 SD 3.47 1.16 Minimum17.0 4.0 Maximum 29.0 8.0 no. subjects 10 10

[0297] Serum Tobramycin Concentrations and Pharmacokinetic Parameters

[0298] Administration of TOBI 300 mg using the PARI LC PLUS deliverysystem produced higher mean serum tobramycin concentrations, a highermean C_(max), and a greater AUC₍₀₋₈₎ than administration of TOBI 60 mgusing the Aerodose delivery system. The mean time to maximum tobramycinconcentration (T_(max)) was similar for the two delivery systems.

[0299] Serum tobramycin concentrations for all subjects were belowquantifiable limits before dosing in both period 1 and period 2. FIGS. 1through 20 graphically illustrate serum tobramycin concentrations beforeand after period 1 and period 2 dosing for all individual subjects.

[0300] After dosing, two subjects had serum tobramycin concentrationsthat could not be measured (i.e., results were below the quantifiablelimit of 0.20 μg/mL) during one of the two treatment periods. These twosubjects were inevaluable for pharmacokinetic analysis during the periodindicated but provided evaluable results for the alternate period.

[0301] Consistent with the high efficiency of the Aerodose system, meanserum tobramycin concentrations were slightly lower throughout the8-hour postdose observation period after delivery of TOBI 60 mg usingthe Aerodose system than after delivery of TOBI 300 mg using the PARI LCPLUS system (Table 15 below). Maximum plasma concentrations for bothregimens were reached within 2 hours after the end of inhalation (TOBI300 mg and PARI inhaler: 1 hr and 2 hr means=0.63 μg/mL; TOBI 60 mg andAerodose inhaler: 2 hr mean=0.48 μg/mL). By 8 hours after the end ofinhalation, the plasma concentrations were below the limit ofquantitation in 5 subjects after the Aerodose inhaler and in twosubjects after the PARI LC PLUS nebulizer. TABLE 15 SERUM TOBRAMYCINCONCENTRATIONS AND PHARMACOKINETIC PARAMETERS Intent to Treat (n = 10)TOBI 300 mg TOBI 60 mg Parameter* PARI LC PLUS^(a) AeroDose^(b) SerumTobramycin (μg/mL): Time Before and After Dosing: Predose 0.00 (0.00) 90.00 (0.00) 9 30 minutes 0.42 (0.24) 9 0.22 (0.23) 9  1 hour 0.63 (0.29)9 0.41 (0.22) 9  2 hours 0.63 (0.25) 9 0.48 (0.20) 9  4 hours 0.50(0.16) 9 0.38 (0.10) 9  8 hours 0.22 (0.14) 9 0.13 (0.12) 9Pharmacokinetic Parameters: C_(max) (μg/mL) 0.677 (0.279) 9 0.482(0.201) 9 T_(max) (hr) 2.213 (0.923) 9 2.207 (0.788) 9 T_(½) (hr) 4.269(1.058) 9 6.071 (3.357) 9 AUC₍₀₋₈₎(μg/mL · hr) 3.622 (1.319) 9 2.553(0.989) 9 AUC_((0-∞))(μg/mL · hr) 5.273 (1.699) 9 4.630 (0.967) 9Pharmacokinetic Parameters Normalized to Dose: C_(max) (μg/mL)/mg 0.002(0.001) 9 0.008 (0.003) 9 AUC₍₀₋₈₎(μg/mL · hr)/mg 0.012 (0.004) 9 0.043(0.016) 9 AUC_((0-∞))(μg/mL · hr)/mg 0.018 (0.006) 9 0.077 (0.016) 9

[0302] Pharmacokinetic Results

[0303] The mean of the maximum tobramycin concentrations for allsubjects (C_(max) in Table 15 above) was greater after TOBI 300 mgdelivered by the PARI LC PLUS system (mean ±SD=0.677±0.279 μg/mL) thanafter TOBI 60 mg delivered by the Aerodose system (0.482±0.201 μg/mL).This mean difference in log C_(max) was statistically significant(p=0.0018), and there was no evidence to suggest the presence of acarryover effect in C_(max) (p=0.6400). The Aerodose inhaler was moreefficient than the PARI LC PLUS nebulizer based on C_(max) resultsadjusted for TOBI dose administered (TOBI 300 mg with PARI LCPLUS=0.002±0.001 (μg/mL)/mg; TOBI 60 mg with Aerodose=0.008±0.003(μg/mL)/mg).

[0304] The time to maximum tobramycin concentrations (T_(max)) wasvirtually identical for the two delivery systems (mean=2.213 hours forPARI LC PLUS and 2.207 hours for Aerodose systems in Table 15 above).T_(max) results in the present study were consistent with observationsin a previous study¹⁵ that peak serum tobramycin concentrations occurredat 1 to 2 hours after inhalation.

[0305] The mean elimination half-life (T_(1/2)) was 4.269 hours for thePARI LC PLUS system and 6.071 hours for the Aerodose system (Table 7).

[0306] The mean area under the serum concentration-time curve through 8hours postdose (AUC₍₀₋₈₎) was significantly greater (p=0.0002 on logAUC₍₀₋₈)) after TOBI 300 mg delivered by the PARI LC PLUS system(3.622±1.319 μg/mL·hr) than after TOBI 60 mg delivered by the Aerodosesystem (2.553±0.989 μg/mL·hr). There was no evidence (p=0.7858) tosuggest the presence of carryover effect in AUC₍₀₋₈₎. The greaterefficiency of the Aerodose inhaler was also seen in dose-normalizedAUC₍₀₋₈₎ results (TOBI 300 mg with PARI LC PLUS=0.012±0.004[μg/mL·hr]/mg; TOBI 60 mg with Aerodose 0.043±0.16 [μg/mL·hr]/mg).

[0307] The mean area under the serum concentration by time curveextrapolated to infinity (AUC_((0-∞)) in Table 7 above) was notsignificantly different (p=0.5477 on log AUC_((0-∞))) afteradministration of TOBI 300 mg using the PARI system (5.273±1.699μg/mL·hr) than after administration of TOBI 60 mg using the Aerodosesystem (4.630±0.967 μg/mL·hr). No carryover effect was detected(p=0.6006). The greater efficiency of the Aerodose inhaler was similarlyseen in dose-normalized AUC_((0-∞)) results (TOBI 300 mg with PARI LCPLUS=0.018±0.006 [μg/mL·hr]/mg; TOBI 60 mg with Aerodose=0.077±0.16[μg/mL·hr]/mg).

[0308] Unplanned, exploratory analyses suggested that female subjectsachieved slightly higher C_(max), AUC₍₀₋₈₎ and AUC_((0-∞)) results thanmale subjects after both TOBI 300 mg and TOBI 60 mg treatments.

[0309] Extent of Exposure

[0310] The duration of exposure to study drug and the dose of study drugwere not varied in this study. All 10 subjects received a single 300 mg(5 mL) TOBI dose using the PARI LC PLUS jet nebulizer with the DeVilbissPulmoAide compressor delivery system (control treatment) on one occasionand a single 60 mg (1 mL) TOBI dose using the Aerodose inhaler(experimental treatment) on a second occasion. Each dose wasradiolabeled with up to 10 MBq ^(99m)Tc-DTPA and administered in arandomized two-way crossover fashion separated by a 44-hour minimumwashout period.

[0311] The mean whole lung deposition using the PARI LC PLUS nebulizerwas 8.2% (24.5 mg) of the 300 mg TOBI dose. The mean whole lungdeposition using the Aerodose inhaler was 34.8% (20.9 mg) of the 60 mgTOBI dose. A mean of 14.4% (43.3 mg) and 31.5% (18.9 mg) of thecorresponding doses were deposited in the oropharynx using the PARI LCPLUS and Aerodose inhalers, respectively. Both inhaler systems wereconfigured such that each subject's exhaled material was collected anddid not escape with radioactive aerosol into the surrounding atmosphere.The PARI LC PLUS nebulizer also included a system to collect anyradiolabeled droplets escaping from the nebulizer.

[0312] Bronchospasm

[0313] In this study, decreases in the relative FEV₁ % predicted ≧10%(not clinically significant if <20%) and ≧20% (clinically significant)from predose measurements to 30-minutes postdose measurements with eachdelivery system were used as indicators of bronchospasm (airwayreactivity). Reductions in FEV₁ % predicted ≧20% were consideredclinically significant for the purposes of the study. No subject had adrop in FEV₁ % predicted ≧10% from predose to postdose regardless ofdelivery system during this study.

[0314] Discussion and Overall Conclusions

[0315] The study of this example demonstrates that the AeroDose™ inhalerwas more efficient in delivery of aerosolized tobramycin to the lungs ofhealthy adult volunteers than the approved PARI LC PLUS jet nebulizerwith DeVilbiss PulmoAide compressor. Since the Aerodose inhaler isbreath-actuated and generates aerosol only during inhalation,proportionally more of the Aerodose dose should be delivered to thelungs than is delivered by the PARI LC PLUS, and there should be minimalwastage of drug by aerosolization during exhalation or by incompleteaerosolization of the contents of the drug reservoir.

[0316] During the study, the Aerodose inhaler delivered a significantlygreater percentage of the dose to the lungs than the PARI LC PLUSnebulizer (mean 34.8% vs. 8.2%: p=0.005). The actual amount of the dosedeposited in the lungs was slightly but not significantly less using theAerodose inhaler than using the PARI LC PLUS nebulizer (20.9 mg vs. 24.5mg: p=0.202). These data demonstrate that the Aerodose inhaler deliverednearly as much tobramycin to the lungs as the PARI LC PLUS nebulizerdespite nebulizing one-fifth the amount of tobramycin.

[0317] Approximately 32% of the Aerodose dose was wasted on the inhalerand exhalation filter combined. By contrast, more than 74% of the PARILC PLUS dose was wasted by deposition on the inhaler and exhalationfilter.

[0318] When the Aerodose inhaler was used, 15.2% (9.1 mg) of the 60 mgTOBI dose remained deposited on the inhaler, and 16.9% (10.1 mg) wasdeposited on the exhalation filter. Since no aerosolization occurredduring exhalation when the Aerodose was used, the observed depositioncould have been due only to seepage through the mouth-inhaler seal or toresidual radiolabeled tobramycin inhaled but immediately exhaled and notdeposited in either the lungs or the oropharynx (including esophagus andstomach). Four subjects were noted to have either experienced problemsmaintaining a seal around the mouthpiece of the Aerodose inhaler orreported that the inhaler failed to nebulize one of the two aliquots ofthe dose solution. These subjects had approximately 47%, 19%, 53%, and26%, respectively, of the 60 mg dose deposited on the inhaler andexhalation filter combined. The highest two of these figures were abovethe range noted for the rest of the subjects (ranging from 17% to 40%deposited on inhaler and exhalation filter combined). Problems withincomplete nebulization or wide variation in subject inhalationeffectiveness may have contributed to the amount of wastage of drugduring Aerodose usage in the present study.

[0319] By comparison, when the PARI LC PLUS jet nebulizer was used,42.6% (127.8 mg) of the 300 mg TOBI dose remained deposited on theinhaler, and 31.6% (94.8 mg) was deposited on the exhalation filter.Presumably, most or all of the exhalation filter deposition was due tocontinued aerosolization and consequent loss of drug while subjectsexhaled.

[0320] Thus, both the Aerodose inhalers and PARI LC PLUS nebulizerswasted drug product in the present study by reason of retention ofradiolabeled drug on or in the inhaler or deposition of drug on theexhalation filter (an average of approximately 19 of 60 mg wasted whenthe Aerodose inhaler was used and approximately 223 of 300 mg wastedwhen the PARI LC PLUS nebulizer was used). The proportion of the totaldose wasted using the Aerodose inhaler was less than half of that wastedusing the approved PARI LC PLUS nebulizer.

[0321] The Aerodose inhaler also appeared to exhibit better “targeting”or delivery of the dose to the lungs, the target site of the usual P.aeruginosa infection in cystic fibrosis patients, than the PARI LC PLUSnebulizer. The Aerodose inhaler deposited slightly more tobramycin inthe lungs than in the oropharynx, esophagus, and stomach (lungs 34.8%vs. 31.5% of the 60 mg dose). By comparison, the PARI LC PLUS nebulizerdeposited proportionally less of the dose in the lungs than inoropharynx, esophagus, and stomach (lungs 8.2% vs. 14.4% of the 300 mgdose). The ratio of lung to oropharyngeal, esophagus, and stomachcombined was approximately 1.1 for the Aerodose inhaler and 0.6 for thePARI LC PLUS nebulizer.

[0322] In addition to greater efficiency by greater delivery of drug tothe lungs and proportionally greater targeting of the lungs, theAerodose inhaler was also anticipated to be more efficient by reason ofproportionally greater delivery of tobramycin to peripheral rather thancentral lung regions. The Aerodose particle MMD is smaller (mean MMD=4.0μm) than that produced by the PARI LC PLUS nebulizer (mean MMD=4.8 μm),so the expectation was that the Aerodose inhaler would deposit a greaterproportion of aerosol generated during inhalation in the peripheralairways than the PARI LC PLUS. During the study, the Aerodose inhalerdeposited 13.2% (7.9 mg) of the 60 mg dose in the peripheral airways,while the PARI LC PLUS nebulizer deposited 3.1% (9.3 mg) in peripheralairways. Although the Aerodose inhaler achieved proportionally greaterperipheral deposition than the PARI LC PLUS nebulizer, both inhalersfell short of amounts predicted for peripheral deposition based ontheoretical considerations (Aerodose estimated to peripherally deposit60% (36 mg) of the 60 mg dose=1.0 mL fill volume·0.95aerosolization·0.62 respirable particles; PARI LC PLUS estimated toperipherally deposit 16% (48 mg) of the 300 mg dose=5.0 ML fillvolume·0.64 aerosolization·0.44 respirable particles).

[0323] Results of the study also showed that the Aerodose inhalerrequired significantly less nebulization time than the PARI LC PLUSnebulizer (mean 20.4 vs. 5.7 minutes, respectively). The 5.7 minuteaverage nebulization time for the Aerodose inhaler did not include theamount of time needed to fill the drug reservoir before nebulization ofthe second aliquot. Based on nebulization time results and other inhalerfeatures including portability, ease of use, and lack of a need for acompressor, it is anticipated that the Aerodose inhaler would improvepatient compliance.

[0324] Serum tobramycin concentrations, maximum concentrations, andextent of absorption were greater after administration of TOBI 300 mgusing the PARI LC PLUS nebulizer than after administration of TOBI 60 mgusing the Aerodose inhaler. These results appeared to be consistent withamounts of tobramycin deposited in lungs and oropharynx (includingesophagus and stomach) combined where systemic absorption occurred (meantobramycin deposited in lungs and oropharynx combined=67.8 mg after TOBI300 mg; mean=39.8 mg after TOBI 60 mg). Mean serum tobramycinconcentrations were higher throughout the 8-hour observation periodafter administration of TOBI 300 mg using the PARI LC PLUS nebulizerthan after administration of TOBI 60 mg using the Aerodose inhaler. MeanC_(max) values were 0.677 and 0.482 μg/mL for TOBI 300 mg and TOBI 60mg, respectively (statistically significant: p=0.0018). Mean T_(max)results for both inhalers were virtually identical (2.213 and 2.207hours, respectively). Apparent absorption of tobramycin wassignificantly greater during the 8-hour postdose period after TOBI 300mg than after TOBI 60 mg (mean AUC₀₋₈=3.622 and 2.553 μg/mL·hr,respectively; statistically significant: p=0.0002), but no treatmentdifferences were noted in AUC_(0-∞) (TOBI 300 mg and TOBI 60 mgmeans=5.273 and 4.630 μg/mL·hr, respectively; p=0.5499).

[0325] Current results suggested that the 60 mg TOBI dose aerosolizedusing the Aerodose inhaler produced tobramycin deposition and serumtobramycin concentration results that were significantly orsubstantially less than results obtained after aerosolization of theapproved TOBI 300 mg dose using the PARI LC PLUS nebulizer. Normalizedfor administered dose, the Aerodose inhaler was substantially moreefficient on a per milligram basis in delivery of tobramycin to thesystemic circulation than the PARI LC PLUS nebulizer. These results areconsistent with the higher deposition (on a milligram basis) in thelung.

[0326] Results of the study also showed that single doses of TOBI 300 mgdelivered using the PARI LC PLUS jet nebulizer and of TOBI 60 mgdelivered using the Aerodose breath actuated nebulizer were safe andwell-tolerated by healthy adult male and female volunteers. No instancesof bronchospasm were observed, and no notable quantitative changes inpulmonary function were seen. No notable adverse events (AEs) werereported by subjects, and there were no apparent differences betweentreatment groups in incidence of any AE. Six treatment emergent AEs werereported by 4 subjects, but all events were mild in intensity. Twoinstances of headache were considered possibly or definitely related totreatment. No clinically significant laboratory results or changes inresults were observed. No adverse vital signs, body weights, physicalfindings, or electrocardiogram results were observed. No evidence ofsystemic toxicity, as measured by unusually high serum tobramycinconcentrations, was observed.

Example 3 In Vivo Study 2

[0327] A comparison was made of the safety, pharmacokinetics, aerosoldelivery characteristics, and nebulization time of the conventional doseand inhalation delivery system (5 mL ampoule containing 300 mgtobramycin and 11.25 mg sodium chloride in sterile water for injection(TOBI® tobramycin solution for inhalation, Chiron Corporation, Seattle,Wash.), pH 6.0; administered with a PARI LC PLUS™ jet nebulizer with aDeVilbiss PulmoAide™ compressor set to deliver an output pressure of 20psi—the “control delivery system”) with a dose of 420 mg TobramycinSolution for Inhalation at 120 mg/mL (excipient 3.5 mL of 1/4 normalsaline adjusted to a pH of 6.0±0.5; 420 mg in 3.5 mL) delivered by thePARI LC PLUS™ jet nebulizer with a Invacare MOBILAIRE™ compressor set todeliver an output pressure of 35 psi (the “experimental deliverysystem”).

[0328] The study was designed as an open label, randomized, single-dose,multicenter, two treatment, active-control, and parallel trial. Eachpatient was administered a single aerosolized dose of study drug witheither the control delivery system or the experimental delivery system.In accordance with the study design, a total of 36 eligible male andfemale patients 12 years of age or older with a confirmed diagnosis ofcystic fibrosis were enrolled with a minimum of 4 patients at each site.A 2:1 randomization ratio was employed for assignment of patients to thetreatment groups. In the presence of the investigator or studycoordinator, each patient was to self-administer either a single dose of300 mg TOBI® with the control delivery treatment or a single dose of 420mg Tobramycin Solution for Inhalation with the experimental deliverytreatment as listed below.

[0329] Control Treatment:

[0330] Aerosolized 300 mg TOBI® was delivered by PARI LC PLUS jetnebulizer/DeVilbiss PulmoAide compressor: Preservative free tobramycinfor inhalation 60 mg/mL (excipient 5 mL of 1/4 normal saline adjusted toa pH of 6.0±0.5); 300 mg in 5 mL; lot number 03K1C (TOBI® at 60 mg/mL).

[0331] Experimental Treatment (420 mg Tobramycin Solution for Inhalationor “TSI”):

[0332] Aerosolized 420 mg Tobramycin Solution for Inhalation (TSI) wasdelivered by PARJ LC PLUS jet nebulizer/Invacare MOBILAIRE compressor:Preservative free tobramycin 120 mg/mL (excipient 3.5 mL of ¼ normalsaline adjusted to a pH of 6.0±0.5); 420 mg in 3.5 mL.

[0333] Both 300 mg TOBI® and 420 mg Tobramycin Solution for Inhalationare sterile, non-pyrogenic, preservative-free antibiotics prepared foraerosolization. Each mL of TOBI® contains 60 mg tobramycin and 2.25 mgsodium chloride in sterile water for injection, pH 6.0±0.5 (controltreatment). Each mL of TSI contains 120 mg tobramycin and 2.25 mg sodiumchloride in sterile water for injection, pH 6.0+0.5 (experimentaltreatment). Drug supplies for this study were manufactured by AutomatedLiquid Packaging (ALP), Woodstock, Ill. All repackaging, labeling, anddistribution for clinical use was provided by Packaging Coordinators,Inc. (PCI), Philadelphia, Pa. Study drug and device supplies wereshipped from Chiron Corporation, Emeryville, Calif. for each patientupon enrollment in the study.

[0334] The duration of study participation for each patient wasapproximately two weeks including a brief (one day one week beforetreatment) screening period, one day treatment period, and a follow-upone-week after treatment. Study treatments were evaluated for safety andaerosol delivery characteristics up to eight hours post-dose on the dayof the single dose treatment administration. The patient was to returnto the clinic for a seven day post-treatment follow-up assessment ofsafety. There were no planned interim safety analyses.

[0335] Criteria for Evaluation:

[0336] Safety:

[0337] Incidence of bronchospasm defined as FEV₁ decrease of ≧10% andFEV₁ decrease of ≧20% from predose to 30 minutes postdose;

[0338] Relative change and absolute change in airway response (FEV₁)after single dose of study drug;

[0339] Laboratory measures of safety (clinical lab tests, spirometrytesting);

[0340] Incidence of treatment emergent adverse events.

[0341] Aerosol Delivery:

[0342] Pharmacokinetic assessment of sputum and serum tobramycinconcentrations;

[0343] Sputum was collected at pre-dose and 15 minutes, 1, 2, 4, and 8hours after dosing;

[0344] Serum was collected at pre-dose and 10 minutes, 1, 2, 4, 6, and 8hours after dosing;

[0345] Nebulization time.

[0346] Statistical methods: All patients who received a dose of studytreatment were evaluated for safety and aerosol deliverycharacteristics.

[0347] Rate of bronchospasm measured by the percent of patientswith >10% and >20% relative decrease in FEV₁% from pre-dose to 30minutes post-dose was summarized and compared between treatments usingthe Fisher's exact test.

[0348] A two sample t-test was used to compare the relative change inFEV₁% from predose to 30 minutes postdose between experimental andcontrol treatments. Summary statistics for relative and absolute changein FEV₁ were tabulated by treatment.

[0349] Sputum and serum area under curve (AUC₀₋₈) and maximumconcentrations (C_(max)) were summarized and analyzed for treatmentdifferences using a general linear model analysis of variance (ANOVA).Pharmacokinetic parameters were calculated using a non-compartmentalmodel. Sputum and serum concentrations were summarized and graphicallyillustrated by treatment.

[0350] Laboratory measures of safety and incidence of treatment-emergentadverse events were summarized and descriptively compared betweentreatments.

[0351] Nebulization time was recorded and summarized for each of the twodelivery treatments.

[0352] Safety Variables

[0353] Aerosol delivery variables were tobramycin concentrations insputum and serum, sputum and serum tobramycin pharmacokineticparameters, and aerosol nebulization time. Safety variables were theincidence and severity of bronchospasm, measured as the number ofpatients experiencing a >10% and a >20% decrease in forced expiratoryvolume in one second (FEV₁) within 30 minutes after dosing (a ≧20%decrease in FEV₁ was considered clinically significant), the incidenceof treatment emergent adverse events (AEs), clinical laboratory testresults, the number of patients with serum tobramycin concentrations ≧4μg/mL, physical examination findings, and vital signs results.

[0354] Primary Aerosol Delivery Variables

[0355] Evaluation of the aerosol delivery characteristics of 420 mgTobramycin Solution for Inhalation at 120 mg/mL delivered by the PARI LCPLUS™/Invacare MOBILAIRE™ delivery system compared to 300 mg TOBI® at 60mg/mL delivered by the FDA-approved PARI LC PLUS™/DeVilbiss PulmoAide™delivery system was based on determination of sputum and serumtobramycin concentrations, calculation of certain sputum and serumpharmacokinetic parameters, and measurement of nebulization time.

[0356] Sputum Tobramycin Concentrations: Sputum samples wereexpectorated by patients from a deep cough and collected before day 1dosing (predose) and at 0.25, 1, 2, 4, and 8 h after the end of thenebulization period. Sputum samples were collected as close as possibleto specified times and were considered to have been drawn on time within±2 minutes for the 15-minute posttreatment collection and within ±10minutes for the 1-, 2-, 4-, and 8-hour posttreatment collections.Samples collected outside these intervals were considered protocoldeviations. A minimum 100 mg sputum (not saliva) sample was collectedbefore the single dose of study treatment to determine the baselinetobramycin concentration. Immediately after dosing, patients rinsedtheir mouths with 30 mL of normal saline, gargled for 5-10 seconds, andexpectorated the rinse. This sequence of post-treatment rinsing wasrepeated for a total of three rinses. Sputum samples were stored at −70°C. or below until analysis. The concentration of tobramycin was analyzedusing reversed-phase high-performance liquid chromatography (HPLC) withultraviolet detection. Patient sputum samples were first liquefied with0.2 N NaOH and diluted with Tris buffer (20.0 g Trizma base/L). Sputumstandard samples were prepared by spiking diluted pooled sputum from CFpatients with tobramycin to final concentrations of 0, 20, 40, 100, 200,400, and 1000 μg/g of sputum. Assay quality control samples wereprepared by spiking diluted pooled sputum to contain 40, 300, and 800μg/g. The internal standard sisomycin (100 μL, 0.15 mg/mL in Trisbuffer) was added to 100 μL of each standard, control, and subjectsample, followed by 400 μL of acetonitrile and 50 μL of2,4-dinitrofluorobenzene (0.17 g/mL). The sample reaction mixtures wereheated in a dry-block heater for 1 h at 80° C. After addition of 600 μLof 60/40 acetonitrile/water (v/v), 50 μL was analyzed by HPLC. Sampleswere injected onto a Waters Nova-Pak® C-18, 3.9×150 mm, 4 μm columnconnected to a Waters HPLC with 600E pump, 486 or 2487 ultravioletdetector (λ_(max)=360 nm) and 717 Plus autosampler. The mobile phaseconsisted of 0.2% acetic acid in acetonitrile (39/61, v/v), pumped at arate of 1.5 mL/min for 5 min, 2.0 mL/min for an additional 9 or 10 min,depending on the length of the run. Waters Millennium-32 C/S LC Software(version 3.20) was used to operate the Waters HPLC instruments as wellas acquire raw data, process, compute, and report the analyticalresults. The ratio of the peak height of tobramycin to the internalstandard sisomycin (PHR) was calculated. The assay was completed in 8runs. Retention time ranges of 4.2 to 4.4 min, and 10.8 to 11.8 min wereobserved for tobramycin and sisomycin, respectively. A linearrelationship existed between PHR and concentration from 20 to 1000 μg/gfor sputum. The regression model was PHR=Bx+A (x=tobramycinconcentration), weighted 1/x. The lower limit of quantitation was 20μg/g. The concentrations of the standard samples were within 97 to 105%of the nominal concentration, with coefficients of variation not higherthan 3.4%. The precision of the assay, as reflected by the CV of thequality control samples, was 2.3%, 2.2% and 2.6%, for the 40, 300, and800 μg/g samples, respectively. The accuracy of the method, reflected bythe interassay recoveries of the quality control samples, was 103%, 99%,and 98% for the 40, 300, and 800 μg/g quality control samples,respectively. Overall, this method exhibited suitable accuracy andprecision for pharmacokinetic analysis.

[0357] Serum Tobramycin Concentrations: Blood samples were collected atpredose and at 0.167, 1, 2, 4, 6, and 8 h after the end of thenebulization period. Samples were collected as close as possible tospecified times and were considered to have been drawn on time within ±2minutes for the 10-minute posttreatment collection and within ±10minutes for the 1-, 2-, 4-, 6-, and 8-hour posttreatment collections.Samples collected outside these intervals were considered protocoldeviations. Serum was harvested and stored at −70° C. or below untilanalysis. Concentrations of tobramycin in serum were analyzed with amodified fluorescence polarization immunoassay (FPIA) method using theAbbott TDx®/TDxFLx® System. Samples were added directly to the dilutionwell of the sample cartridge. The net polarization was acquired by theTDx®/TDxFLx® apparatus and manually entered into an Oracle database. Aweighted four parameter logistic equation was used to calculate theconcentrations of tobramycin. The concentrations of tobramycin werereported in terms of free base equivalents. For assaying the subjectsamples of the study, calibration standards (0.050, 0.100, 0.200, 0.400,0.600, 0.800, 1.000 μg/mL) and quality control samples (0.150, 0.400,and 0.750 μg/mL) were prepared in house. The assay was completed in 8runs. A linear relationship existed between polarization response andconcentration from 0.050 μg/mL to 1.00 μg/mL. The lower limit ofquantitation was 0.050 μg/mL. The precision of the assay, as reflectedby the CV of the quality control samples, was 3.3%, 4.9%, and 4.9% forthe 0.150, 0.400, and 0.750 μg/mL samples, respectively. The accuracy ofthe method, reflected by the mean interassay recoveries of the qualitycontrol samples, was 101%, 103%, and 104% for the 0.150, 0.400, and0.750 μg/mL samples, respectively. Overall, this method exhibitedsuitable accuracy and precision for pharmacokinetic analysis.

[0358] Nebulization Time:

[0359] Nebulization time was defined as the length of time from thestart of the patient's first tidal breath to completion of aerosoladministration. Aerosol administration was complete when the nebulizerbegan to sputter. If aerosol administration was interrupted for anyreason, the time of interruption and start and stop times of continuedaerosol administration were recorded. If dosing was interrupted,nebulization time was considered to be not calculable.

[0360] Residual Tobramycin in the Nebulizer: The amount of residualtobramycin solution remaining in the nebulizer after completion ofaerosol administration was determined by recording pretreatment andposttreatment weight of the nebulizer system including nebulizer, filtervalve, and study drug. The research coordinator collected residual studydrug remaining in the nebulizer after aerosol administration into a viallabeled with patient information. The vial was returned for measurementof the amount of drug output from the nebulizer and for determination ofthe extent of the concentration of study drug left in the nebulizer.

[0361] Safety Variables

[0362] Bronchospasm: The study protocol prospectively identifiedbronchospasm as an adverse airway response to inhalation of aerosolizedantibiotic of particular relevance to patients with cystic fibrosis. Inorder to determine whether current study treatments producedbronchospasm, patients performed spirometry (pulmonary function) teststo measure FEV₁ before and 30 minutes following completion of studytreatment administration according to the method described in theprotocol. Airway response to the study drug was assessed by evaluatingthe relative percent change in FEV₁ from predose to 30 minutes after theend of treatment using the following formula.${{relative}\quad {FEV}_{1}\quad \% \quad {change}} = {\frac{\begin{matrix}{{30\quad \min \quad {postdose}\quad {FEV}_{1}} -} \\{{predose}\quad {FEV}_{1}}\end{matrix}}{{predose}\quad {FEV}_{1}} \times 100\%}$

[0363] Bronchospasm was defined as a decrease in FEV₁ of ≧10% at 30minutes after dosing, relative to the predose result. A decrease in FEV₁of ≧20% was considered to represent clinically significant bronchospasm.Moreover, if there was a posttreatment decrease in FEV₁ of ≧30%,spirometry was to be repeated until the FEV₁ decrease was <10% below thepredose result. An FEV₁ % decrease ≧30%, and all symptoms associatedwith the change in pulmonary function, were to be recorded as adverseevents. The protocol defined the severity of decrease in FEV₁ based inpart on the National Cancer Institute (NCI) Common Toxicity CriteriaAdverse Events Grading Scale. However, slight inconsistencies in theprotocol definitions of bronchospasm and of the severity of FEV₁ changeswere noted during preparation of the analyses and report. To resolve thedifferences, the actual system used during the analysis to classify theseverity of FEV₁ changes relative to the predose result is listed below.TABLE 16 AIRWAY RESPONSE (FEV₁) (BRONCHOSPASM) FEV₁ % DECREASE BELOWPREDOSE VALUE Severity Protocol Classification Analysis ClassificationMild: ≧10%-≦20% ≧10%-<20% Moderate: >20%-≦30% ≧20%-<30% Severe: >30%≧30%

[0364] Clinical Laboratory Tests

[0365] At screening, laboratory tests were performed to measure serumcreatinine, blood urea nitrogen (BUN), urine protein (proteinuria bydipstick), and to detect pregnancy in females of childbearing potential.If abnormal at screening, serum creatinine, BUN, and urine protein testswere to be repeated before the time of dosing. Final test results wereobtained based on specimens drawn at the follow-up visit on day 8 of thestudy.

[0366] After the mean body weight difference between treatment groupsbecame known by Chiron personnel, estimated creatinine clearance wascalculated for patients using the Cockroft-Gault equation below toevaluate renal clearance characteristics of the two groups and toclarify the pharmacokinetic results of the study.

[0367] Male patients:

[0368] estimated creatinine

[0369] clearance (mL/min) (140-age[yr])(body weight[kg])/72*(serumcreatinine [mg/dL])

[0370] Female patients:

[0371] estimated creatinine

[0372] clearance (mL/min)=0.85*((140-age[yr])(body weight[kg])/72*(serumcreatinine [mg/dL])

[0373] All abnormal laboratory test results, whether present on entryinto the study or arising during the study, were evaluated by the studyinvestigator for clinical significance and relationship to study drug.If the abnormal result was considered unrelated to study drug, theinvestigator was to identify the probable cause of the result.Laboratory results considered markedly abnormal and clinicallysignificant were BUN>16 mmole/l (>45 mg/dl), serum creatinine >177μmole/l (>2 mg/dl), and proteinurea≧3+.

[0374] Other Safety Variables

[0375] Serum assay results were screened for tobramycinconcentrations >4 μg/mL from specimens collected from 10 minutes through8 hours after completion of study treatments. In parallel, patientrecords and CRFs were examined for evidence of systemic toxicitypotentially related to elevated tobramycin levels. Assay results werenot available until after patients' discharge from the study, soscreening for unusually high serum tobramycin concentrations andevidence of systemic toxicity was undertaken when all pertinent resultswere received.

[0376] Pharmacokinetics

[0377] Pharmacokinetic parameters for both sputum and serum tobramycinwere derived to characterize aerosol delivery capabilities of controland experimental treatments. The concentration (C) versus time (t) data(Listings 16.2.5.2 and 16.2.5.3) were analyzed by model-independentmethods to obtain the pharmacokinetic parameters. The areas under theplasma concentration-time curve from time zero (predose) to infinity(AUC) and under the first moment of the plasma concentration-time curve(AUMC) were obtained by the trapezoidal rule, extrapolated to infinity.The terminal rate constant (λ_(z)) was determined by log-linearregression of the terminal phase. The maximum concentration (C_(max))and the time to maximum after the end of the nebulization period(t_(max)) were obtained by inspection. In addition, the followingparameters were calculated:

t _(1/2) =ln(2)/λ_(z)

CL/F=D/AUC

V _(z) /F=CL _(po)/λ_(z)

[0378] where t_(1/2) is the terminal half-life, CL/F is the total bodyclearance, and Vz/F is the terminal volume of distribution.^(i) Sincethe absolute bioavailability of tobramycin (F) in the two formulationsused in this study is not known, the calculated clearance and volume ofdistribution are hybrid parameters that do not account for differencesin bioavailability between the two formulations. All parameters werecalculated for serum; only AUC, C_(max), t_(max), λ_(z), and t_(1/2)were calculated for sputum.

[0379] Concentrations below the lower limit of quantitation were treatedas zero for all calculations. Since there was an insufficient volume ofmatrix to assay tobramycin in the following time points, they wereexcluded from the pharmacokinetic analysis: TABLE 17 EXCLUSIONS FROMPHARMACOKINETIC ANALYSIS Matrix Subject Time Serum 01-110 6 02-116 103-102 0.167, 1, 2 03-105 0, 0.167, 1, 2, 4, 6, 8 03-131 0.167 05-125 4,8 06-120 2 Sputum 08-127 2

[0380] Data Handling

[0381] Case report form data were entered in duplicate into a Clintrial™database by the department of Biostatistics and Clinical Data Management(BCDM) at Chiron Corporation. Data quality assurance was performed usingPL/SQL and SAS™ 6.12 or higher software (SAS Institute, Cary, N.C.).Analysis was performed by Chiron Corporation, using SAS version 6.12 orhigher software, based on a predefined analysis plan developed by ChironCorporation. The estimated overall database error rate was 0.xx % withan upper 95% confidence limit of 0.xx %. This upper confidence limit isbelow the departmental standard of 0.5%.

[0382] Statistical Methods and Determination of Sample Size

[0383] Statistical and Analytical Plans: Serum and sputumpharmacokinetic parameters, the incidence of bronchospasm, and therelative change from predose in 30-minute postdose FEV₁ % predicted wereanalyzed statistically to assess the significance of any apparentdifferences between test and reference treatments. All statistical testsdescribed in following sections were two-tailed tests of significance,and the criterion for statistical significance was set at α=0.05 unlessotherwise noted.

[0384] Primary Aerosol Delivery Analyses:

[0385] All patients who received the single dose of test or referencetreatment were included in the analysis and evaluation of aerosoldelivery characteristics. Aerosol delivery was characterized on thebasis of serum and sputum tobramycin concentrations, derived serum andsputum pharmacokinetic parameters, and nebulization time. The effect oftreatment (300 mg TOBI vs 420 mg TSI), gender, and age group (less than18, 18 years or older) on the AUC, C_(max), λ_(z), CL/F, and Vz/F oftobramycin in serum, and on the AUC, C_(max), and λ_(z) of tobramycin insputum, was analyzed by a three-way analysis of variance. Furthermore,the relationship between body weight and AUC, C_(max), CL/F, and Vz/F oftobramycin in serum, and between body weight and AUC and C_(max) oftobramycin in sputum were analyzed by regression analysis. All testsemployed a significance level α=0.05. All parameters are expressed asthe mean ±SD. A harmonic half-life was estimated as:

{overscore (t _(1/2))}=ln(2)/{overscore (λ_(z))}

[0386] in which {overscore (λ_(z) )} is the arithmetic mean of theterminal rate constants at each dose. The standard deviation of theharmonic half-life, SD({overscore (t_(1/2))}), was obtained as:${{SD}\left( \overset{\_}{t_{1/2}} \right)} = {\frac{\ln (2)}{\overset{\_}{\lambda_{z}}} \times \frac{{SD}\left( \overset{\_}{\lambda_{z}} \right)}{\overset{\_}{\lambda_{z}}}}$

[0387] where SD({overscore (λ_(z))}) is the standard error of the meanterminal rate constant at each dose.

[0388] Safety Analyses

[0389] Analysis of Airway Response:

[0390] The primary safety variable was the rate of bronchospasm, definedas a ≧10% decrease in FEV₁ from predose to 30 minutes after treatment onday 1 of the study. Secondary safety variables were (a) the rate ofclinically significant bronchospasm, defined as a ≧20% decrease in FEV₁from predose to 30 minutes after treatment on day 1, and (b) therelative change in FEV₁ from predose to 30 minutes after treatment onday 1. The rates of occurrence of all instances of bronchospasm (FEV₁ %decrease≧10%) and of all instances of clinically significantbronchospasm (FEV₁ % decrease≧20%) were analyzed to assess thestatistical significance of test vs. reference treatment differencesusing the Fisher's Exact test. The protocol specified that the treatmentdifference in the incidence of bronchospasm would be tested forstatistical significance using the Cochran-Mantel-Haenszel test. Due tothe low incidence of bronchospasm in the enrolled patients, the Fisher'sexact test was used for this analysis since it makes no assumptionsregarding the minimum expected cell frequencies. The test vs. referencetreatment difference in mean relative change from predose in 30-minutepostdose FEV₁ % predicted was tested for statistical significance usingthe two-sample t-test.

[0391] Adverse Events: The total incidence of individual treatmentemergent adverse events (percent of patients who experienced the eventat least once during or after study treatment) was evaluateddescriptively for any noteworthy differences between test and referencetreatments. AEs were also summarized by severity (mild, moderate,severe) and drug relationship (unrelated, possibly related) for test andreference treatments.

[0392] Disposition of Subjects

[0393] A total of 40 patients were screened for the study by the eightinvestigators. Thirty-eight of the 40 screened patients met entrancecriteria, were enrolled in the study (Table 18), and were randomized toone of the two treatments. Enrollment and randomization of the 38patients at the eight sites was as summarized in Table 18 below: TABLE18 ENROLLMENT AND RANDOMIZATION BY SITE AND TREATMENT 300 mg TOBI ® 420mg TSI PARI LC PLUS ™/ PARI LC PLUS ™/ DeVilbiss Invacare PulmoAide ™MOBILAIRE ™ Delivery System Delivery System (no. patients enrolled (no.patients enrolled Site and randomized) and randomized) 01 2 2 02 1 3 032 6 04 0 2 05 2 4 06 2 3 07 2 2 08 3 2 Total enrolled 14 24 andrandomized

[0394] Two of the 40 screened patients failed to meet entrance criteriaand were not enrolled in the study: one patient did not meet theprotocol inclusion criterion requiring patients to have screening FEV₁ %predicted results that were ≧25%; and one patients did not meet theexclusion criterion requiring patients to have not taken inhaled orintravenous aminoglycosides within seven days before study treatmentadministration. Thirty-eight patients met all study entry criteria andwere randomized to treatments. Thirty-seven of the 38 randomizedpatients received one dose of study treatment (Table 18). One patientwas enrolled and randomized but was withdrawn from the study beforedosing due to staff inability to establish venous access for predose day1 (visit 2) blood draws. The 37 randomized and dosed patientsconstituted the intent to treat (ITT) population. All 37 patients whoreceived study treatments completed the study.

Aerosol Delivery Evaluation

[0395] Data Sets Analyzed

[0396] All 37 patients in the ITT population (i.e., those who wererandomized and received a dose of study treatment) were evaluable forthe aerosol delivery objective of the protocol. Twenty four patientsreceived a dose of 420 mg TSI using the PARI LC PLUS™/InvacareMOBILAIRE™ Delivery System, and 13 patients received a dose of 300 mgTOBI® using the PARI LC PLUS™/DeVilbiss PulmoAide™ Delivery System.Patient 08/137 was excluded from all aerosol delivery evaluations due towithdrawal from the study before dosing.

[0397] Demographic and Other Baseline Characteristics

[0398] Demographic Characteristics:

[0399] Nineteen male and 18 female patients, 12 to 44 years of age anddiagnosed with cystic fibrosis, constituted the ITT population.Thirty-one patients were Caucasian, four patients were Hispanic, and twopatients were black. Gender and race distributions were similar betweenthe 420 mg TSI and 300 mg TOBI® treatment groups. On the average, ITTpatients in the 300 mg TOBI® group were approximately 2.7 years older,4.9 centimeters taller, and 10.7 kilograms heavier at screening(visit 1) than ITT patients in the 420 mg TSI group. A similar treatmentdifference in mean body weight was apparent before day 1 (visit 2)dosing, and no noteworthy change in mean weight was noted betweenscreening and day 1.

[0400] Analysis of Aerosol Delivery

[0401] Primary Aerosol Delivery Analysis: Examination of the mean plasmaconcentration-time plot for both formulations in serum (FIG. 10)indicates that tobramycin is rapidly absorbed: all subjects achievedmaximum concentrations in the time span of 10 min to 4 h. An eliminationphase was also observed in the concentration-time profiles, withindividual estimates of half-life ranging from 1.1 to 6.8 h. In sputum(FIG. 11), maximum concentrations were achieved between 15 min and 2 h,and individual estimates of half-life ranged from 0.48 to 9.47 h. Theseestimates are consistent with previous studies.

[0402] Serum and sputum pharmacokinetic parameters are summarized inTables 19 and 20 as follows. TABLE 19 SERUM PHARMACOKINETIC PARAMETERS(MEAN ± SD) OF TOBRAMYCIN AFTER ADMINISTRATION OF 300 MG TOBI AND 420 MGTSI Parameter 300 mg TOBI 420 mg TSI AUC (μg h/mL) 4.38 ± 1.97 4.41 ±1.69 C_(max) (μg/mL) 0.861 ± 0.344 0.906 ± 0.542 Median t_(max) (h) 1(1-4)* 1 (0.17-2) λ_(z) (h⁻¹) 0.250 ± 0.052 0.243 ± 0.098 t_(½) (h) 2.78± 0.58 2.86 ± 1.15 CL/F (L/h) 88 ± 62 114 ± 59  V_(Z)/F (L) 379 ± 325511 ± 278

[0403] TABLE 20 SPUTUM PHARMACOKINETIC PARAMETERS (MEAN ± SD) OFTOBRAMYCIN AFTER ADMINISTRATION OF 300 MG TOBI AND 420 MG TSI Parameter300 mg TOBI 420 mg TSI AUC (μg h/g) 1521 ± 845  1176 ± 686  C_(max)(μg/g) 930 ± 795  935 ± 1040 Median t_(max) (h) 0.25 (0.25-2)* 0.25(0.25-0.25) λ_(z) (h⁻¹) 0.59 ± 0.31 0.52 ± 0.37 T_(½) (h) 1.17 ± 0.981.33 ± 0.95

[0404] The serum and sputum concentration-time curves for bothtreatments were virtually superimposable (FIGS. 10 and 11; Tables 19 and20). Serum parameters (C_(max), t_(max), AUC, CL/F, Vz/F) showed nostatistically significant differences between the treatment groups(Table 19). Sputum parameters (AUC, C_(max), and λ_(z)) also showed nostatistically significant treatment differences (Table 20). Neither agenor body weight had a statistically significant effect on thesepharmacokinetic parameters. In addition, there were no statisticallysignificant correlations between serum and sputum AUC, and between serumand sputum C_(max). The variability of the pharmacokinetic parameters inserum and in sputum was similar to previous trials. In summary, thesefindings indicate that it is possible to achieve comparable serum andsputum levels of tobramycin to the 300 mg TOBI® formulation by using the420 mg TSI formulation.

[0405] Secondary Aerosol Delivery Analyses

[0406] Nebulization Time: Nebulization time was substantially reducedduring administration of the test 420 mg TSI formulation below thatobserved during administration of the marketed 300 mg TOBI® formulation.Mean ±SD total nebulization time was 9.7±3.0 minutes during 420 mg TSIadministration compared to 18.1±3.6 minutes during 300 mg TOBI®administration (Table 21). These findings indicate that the reducednebulization times used in the 420 mg TSI treatment did not change thepharmacokinetics of tobramycin relative to the marketed 300 mg TOBI®formulation. TABLE 21 MEAN (SD) NEBULIZATION TIME 300 mg TOBI 420 mg TSIPARI LC PLUS^(a) PARI LC PLUS^(b) Parameter PulmoAide CompressorMOBILAIRE Compressor [mean (SD)] (n = 13) (n = 24) Nebulization 18.1(3.6) 9.7 (3.0) Time (min) No. pts with data 12 23

[0407] Nebulizer Weight: Nebulizer weight changes from before to afterdosing indicated that the test 420 mg TSI formulation delivered lessproduct to patients than the marketed 300 mg TOBI® formulation. Mean ±SDamounts of product delivered to patients was 1.86±0.53 gm during 420 mgTSI administration and 2.74±1.64 gm during 300 mg TOBI® administration(Table 14.2.2.2), as summarized in Table 11.4-4 below. These findingslikely reflect the smaller 3.5 mL volume of TSI formulation in thenebulizer compared to the 5 mL volume of the TOBI® formulation. TABLE 22MEAN (SD) NEBULIZER WEIGHT AND CHANGE IN WEIGHT 300 mg TOBI^(a) 420 mgTSI^(b) PARI LC PLUS PARI LC PLUS Parameter PulmoAide CompressorMOBILAIRE Compressor [mean (SD)] (n = 13) (n = 24) Nebulizer Weight (gm)Predose 68.25 (7.30) 69.17 (0.61) No. patients with 13 24 data Postdose65.51 (6.89) 67.30 (0.80) No. patients with 13 23 data Change in weight−2.74 (1.64)^(c) −1.86 (0.53) No. patients with 13 23 data

[0408] Discussion

[0409] Aerosol delivery findings indicate that it is possible to achievecomparable serum and sputum levels of tobramycin to the 300 mg TOBI®formulation by using the 420 mg TSI formulation. Present findings alsoindicate that the reduced nebulization times and reduced amount ofproduct delivered to patients during administration of the 420 mg TSItreatment did not change the pharmacokinetics of tobramycin relative tothe marketed 300 mg TOBI® formulation.

[0410] Mean serum tobramycin concentration-time plots for bothformulations indicate that tobramycin is rapidly absorbed: all subjectsachieved maximum concentrations in the time span of 10 min to 4 h. Anelimination phase was also observed in the concentration-time profiles,with individual estimates of half-life ranging from 1.1 to 6.8 h. Insputum, maximum concentrations were achieved between 15 min and 2 h, andindividual estimates of half-life ranged from 0.48 to 9.47 h.

[0411] The serum and sputum concentration-time curves for bothtreatments in the present study were virtually superimposable. Serumparameters (C_(max), t_(max), AUC, CL/F, Vz/F) showed no statisticallysignificant differences between the treatment groups. Mean (+SD) serumC_(max) results for both the 420 mg TSI and the 300 mg TOBI® groups(0.906±0.542 μg/mL vs. 0.861±0.344 μg/mL, respectively) were consistentwith results from previous studies.^(5,40,41) The average serumconcentration of tobramycin one hour after inhalation of a single 300 mgdose of TOBI® by CF patients was 0.95 μg/mL.⁵ After 20 weeks of therapyon the TOBI® regimen, the average serum tobramycin concentration onehour after dosing was 1.05 μg/mL.

[0412] Sputum parameters (AUC, C_(max), and λ_(z)) also showed nostatistically significant treatment differences in the present study.Mean (±SD) sputum C_(max) results for both the 420 mg TSI and the 300 mgTOBI® groups (935±1040 μg/g vs. 930±795 μg/g, respectively) wereconsistent with results from previous studies.^(5,40,41) Sputum resultsin the present study were highly variable. By comparison, highvariability of tobramycin concentration in sputum was also observed inboth Phase 3 trials.^(29,30) Ten minutes after inhalation of the first300 mg dose of TOBI® in the Phase 3 trials, the average concentration oftobramycin in sputum was 1237 μg/g (ranging from 35 to 7414 μg/g).Tobramycin does not accumulate in sputum; after 20 weeks of therapy withthe TOBI® regimen, the average concentration of tobramycin at tenminutes after inhalation was 1154 μg/g (ranging from 39 to 8085 μg/g).Two hours after inhalation, sputum concentrations declined toapproximately 14% of the tobramycin levels measured at ten minutes afterinhalation.

[0413] Neither age nor body weight had a statistically significanteffect on serum and sputum pharmacokinetic parameters. In addition,there were no statistically significant correlations between serum andsputum AUC and between serum and sputum C_(max).

[0414] Nebulization time for the test 420 mg TSI formulation wassubstantially reduced below that observed during administration of themarketed 300 mg TOBI® formulation (mean ±SD=9.7±3.0 min vs. 18.1±3.6min, respectively). Nebulization times for the marketed 300 mg TOBI®formulation were consistent with previous studies. 40,41 Therefore, thestudy achieved a key benchmark with the demonstration that thealternative delivery system, consisting of 3.5 mL of a 120 mg/mL (total420 mg tobramycin) Tobramycin Solution for Inhalation (TSI) deliveredusing a PARI LC PLUS™ jet nebulizer driven by an Invacare MOBILAIRE™compressor, reduced nebulization time below 10 minutes on the average.

[0415] Finally, present findings indicate that the reduced nebulizationtimes during administration of the 420 mg TSI treatment did not changethe pharmacokinetics of tobramycin relative to the marketed 300 mg TOBI®formulation.

[0416] Safety findings indicate that both a single dose of the 420 mgTSI formulation and a single dose of the marketed 300 mg TOBI®formulation were well-tolerated by patients with cystic fibrosis. Theincidence of bronchospasm (>10% relative decrease in FEV₁) wasapproximately 8% for each treatment (two 420 mg TSI and one 300 mg TOBI®patients); a single patient in the 300 mg TOBI® group had clinicallysignificant bronchospasm (≧10% relative decrease in FEV₁). The treatmentmean relative decrease in FEV₁ was −3.36±5.47% for 420 mg TSI and−2.14±9.62% for 300 mg TOBI®.

[0417] By comparison, in the Phase III trials of TOBI®, the medianchange in FEV₁ 30 minutes after the first dose of study drug had beenadministered was −1.8% in the tobramycin group. At Week 20, the medianchange in FEV₁ was −2.0% in the tobramycin group. Because up to 95% ofCF patients have bronchodilator-responsive airflow obstruction, and thewithin-subject variability for pulmonary function tests in CF patientshas been documented to be greater than in normal patients, a ≧20%decrease in FEV₁ was considered clinically significant.³³ Twelve of 258TOBI® patients (4.7%) had a >20% decrease in FEV₁ with TOBI®administration. Only two of these patients documented acute symptoms,and no patients had a ≧20% decrease in FEV₁ more than once with TOBI®.

[0418] The present study also showed that the incidence of othertreatment-related adverse events was very low (2 of 24 TSI patients and1 of 13 TOBI® patients=8%) and did not differ between treatments. Allthree patients reported mild to moderate decreased pulmonary functiontest results, and one of the three patients also reported severe cough.Among all treatment-emergent AEs, events reported most frequently by 420mg TSI patients were cough (4 patients 17%), crepitations and sorethroat (13%), and pyrexia, nasal congestion, rhinorrhoea, and sputumincreased (8% ). AEs reported most frequently by 300 mg TOBI® patientswere cough (3 patients=23%) and sore throat, dyspnoea, and rhinorrhoea(15%). These events were mostly mild to moderate in intensity (twoinstances of severe cough), were most likely related to patientsunderlying cystic fibrosis and other medical conditions, and wereconsistent with previous large Phase 3 study results.^(29,30) A singlepatient experienced serious non-drug-related symptoms (SAEs) indicativeof an exacerbation of CF. None of the patients in the study werewithdrawn due to AEs, and no other clinically significant findings werenoted in physical examinations, vital signs, or other safetymeasurements that represented an increase in risk to patients by reasonof administration of study treatments.

CONCLUSIONS

[0419] The findings of the present study indicate that it is possible toachieve comparable serum and sputum levels of tobramycin to the 300 mgTOBIG® formulation by using 420 mg TSI formulation. Current findingsalso indicate that the reduced nebulization times used in the 420 mg TSItreatment did not change the pharmacokinetics of tobrarnycin relative tothe marketed 300 mg TOBI® formulation. Mean plasma concentration-timeplots for both formulations in serum indicate that tobramycin is rapidlyabsorbed: all subjects achieved maximum concentrations in the time spanof 10 min to 4 h. An elimination phase was also observed in theconcentration-time profiles, with individual estimates of half-liferanging from 1.1 to 6.8 h. In sputum, maximum concentrations wereachieved between 15 min and 2 h, and individual estimates of half-liferanged from 0.48 to 9.47 h. These estimates are consistent with previousstudies.

[0420] The serum and sputum concentration-time curves for bothtreatments were virtually superimposable. Serum parameters (C_(max),t_(max), AUC, CL/F, Vz/F) showed no statistically significantdifferences between the treatment groups. Sputum parameters (AUC,C_(max), and λ_(z)) also showed no statistically significant treatmentdifferences. Neither age nor body weight had a statistically significanteffect on these pharmacokinetic parameters. In addition, there were nostatistically significant correlations between serum and sputum AUC, andbetween serum and sputum C_(max).

[0421] During administration of the test 420 mg TSI formulation,nebulization time was substantially reduced below that observed duringadministration of the marketed 300 mg TOBI® formulation (mean±SD=9.7±3.0 min vs. 18.1±3.6 min, respectively). The apparent treatmentdifference in change in nebulizer weight likely reflected the differentstarting volumes of TSI and TOBI® formulations in the nebulizer (mean±SD=1.86±0.53 g vs. 2.74±1.64 g, respectively).

[0422] Aerosol delivery findings indicate that it is possible to achievecomparable serum and sputum levels of tobramycin to the 300 mg TOBI®formulation by using the 420 mg TSI formulation. Current findings alsoindicate that the reduced nebulization times during administration ofthe 420 mg TSI treatment did not change the pharmacokinetics oftobramycin relative to the marketed 300 mg TOBI®formulation.

[0423] While the preferred embodiments of the invention have beenillustrated and described, it will be appreciated that various changescan be made therein without departing from the spirit and scope of theinvention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of thetreatment of a patient in need of such treatment comprisingadministering to the patient a dose of 4.0 ml or less of a nebulizedaerosol formulation comprising from about 60 to about 200 mg/ml of anaminoglycoside antibiotic in less than 10 minutes.
 2. A method of claim1 wherein the dose comprises less than about 3.75 ml of the nebulizedaerosol formulation.
 3. A method of claim 1 wherein the dose comprises3.5 ml or less of the nebulized aerosol formulation.
 4. A method ofclaim 1 wherein the aerosol formulation comprises from about 80 to about180 mg/ml of the aminoglycoside antibiotic.
 5. A method of claim 1wherein the aerosol formulation comprises from about 90 to about 150mg/ml of the aminoglycoside antibiotic.
 6. The method of claim 1 whereinthe aminoglycoside antibiotic is selected from the group consisting ofgentamycin, amikacin, kanamycin, streptomycin, neomycin, netilmicin andtobramycin.
 7. A method of claim 6 wherein the aminoglycoside antibioticis tobramycin.
 8. A method of claim 7 wherein the dose comprises 3.5 mlor less of a nebulized aerosol formulation comprising from about 80 toabout 180 mg/ml of tobramycin.
 9. A unit dose device, comprising acontainer containing less than about 4.0 ml of an aminoglycosideantibiotic formulation comprising from about 60 to about 200 mg/ml of anaminoglycoside antibiotic in a physiologically acceptable carrier.
 10. Aunit dose device of claim 9 which contains less than about 3.75 ml ofthe aminoglycoside antibiotic formulation.
 11. A unit dose device ofclaim 9 which contains 3.5 ml or less of the aminoglycoside antibioticformulation.
 12. A unit dose device of claim 9 wherein theaminoglycoside antibiotic formulation comprises from about 80 to about180 mg/ml of the aminoglycoside antibiotic.
 13. A unit dose formulationof claim 9 wherein the aminoglycoside antibiotic formulation comprisesfrom about 90 to about 150 mg/ml of the aminoglycoside antibiotic.
 14. Aunit dose formulation of claim 9 wherein the aminoglycoside antibioticis selected from the group consisting of gentamycin, amikacin,kanamycin, streptomycin, neomycin, netilmicin and tobramycin.
 15. A unitdose formulation of claim 14 wherein the aminoglycoside antibiotic istobramycin.
 16. A unit dose device of claim 9 which contains less thanabout 4.0 ml of aminoglycoside antibiotic formulation comprising fromabout 80 to about 180 mg/ml of tobramycin.
 17. A system for deliveringan aminoglycoside antibiotic formulation to a patient in need of suchtreatment, comprising a unit dose device comprising a containercontaining less than about 4.0 ml of an aminoglycoside antibioticformulation comprising from about 60 to about 200 mg/ml of anaminoglycoside antibiotic in a physiologically acceptable carrier, andmeans for delivering the aminoglycoside antibiotic formulation from theunit dose device for inhalation by the patient in aerosolized form inless that 10 about minutes.